Disease-site-specific liposomal formulation

ABSTRACT

The present invention provides a clinically applicable, safe and convenient, pharmaceutical composition for disease site-specific treatment. The pharmaceutical composition for disease site-specific treatment comprises a stealth liposome having a prostaglandin I2 receptor agonist encapsulated therein.

TECHNICAL FIELD

The present invention relates to a disease-site-specific liposomalformulation.

BACKGROUND ART

Compound (A) (ONO-1301) is a low-molecular-weight compound having bothPGI2 receptor (IP) agonism and thromboxane (TX) A2 synthase inhibitoryactivity. Compound (A), which has PGI2 agonistic activity, is known tobe useful for prevention and/or treatment of thrombosis,arteriosclerosis, ischemic heart disease, gastric ulcer, hypertension,etc. (Patent Literature (PTL) 1).

On the other hand, prostaglandin (PG) I2 receptor (IP) agonists,prostaglandin EP2 agonists, and prostaglandin EP4 agonists, such asONO-1301, can be used as endogenous repair factor production promotersfor many diseases at low doses by inducing many body regenerationfactors, such as a hepatocyte growth factor (HGF), a vascularendothelial cell growth factor (VEGF), a stromal cell-derived factor(SDF-1), and a high-mobility group box protein 1 (HMGB1); and theseagonists are known to be useful as regenerative therapies (PatentLiterature (PTL) 2).

However, since there are concerns about side effects of compound (A),such as diarrhea following oral administration, and vasodilation andhypotensive effects in intravenous administration, the development of adosage form that is capable of preventing exposure to a highconcentration in the gastrointestinal tract or a rapid increase of theblood concentration, placing less burden on the patient, and maximizingdrug efficacy is strongly desired. In the development of long-termsustained-release injectable formulations, many studies have beenconducted on methods for controlling drug release by microspheres(hereinafter sometimes abbreviated as MS) with an average particlediameter of about 30 μm, containing a drug and using a poorlywater-soluble polymer. A biodegradable polymer is used as the polymer sothat the base does not remain at the site of administration after drugrelease. In particular, for example, polylactic acid polymers(hereinafter sometimes abbreviated as PLA) and lactic acid-glycolic acidcopolymers (hereinafter sometimes abbreviated as PLGA), which have beenused in surgical sutures, bone-fixing bolts, etc., are used. Thesebiodegradable polymers are used in the LH-RH derivative injectableformulation Leuplin (sold by Takeda Pharmaceutical Co., Ltd.) and thelong-acting somatostatin derivative Sandostatin LAR (sold by NovartisPharmaceuticals).

Drugs used in microspheres include bioactive peptides, various hormones,growth factors, antibodies, peptides such as genes and various cellgrowth/differentiation inducing factors, proteins, nucleic acids, andthe like. Compound (A) (Patent Literature (PTL) 3), and compound (B) andcompound (C) (Patent Literature (PTL) 4) are known as low-molecularcompounds.

These drugs can be administered, for example, by intramuscularadministration, subcutaneous injection, or patch application to variousorgans, of an MS formulation; in a dosage form that can continuouslymaintain the drug concentration in the tissue at a disease site, or in adosage form that can maintain the blood concentration, such asintravenous infusions. When administered at a disease site, theseformulations for administration maintain a high drug concentration inthe vicinity of an administration site, and exhibit intravenousinfusion-like blood kinetics; and do not have a drug delivery system(DDS) effect, which is an effect of accumulating a drug at a diseasesite. Specifically, DDS is a technique for delivering a required amountof a drug to a required place at a required time.

On the other hand, there is a known lung disease site-specifictherapeutic agent whose mechanism is such that intravenous injection ofa small amount of an MS formulation accumulates the MS formulation inthe lungs and allows gradual release of a drug in the lungs, therebymaintaining a high concentration of the drug in the lungs (PatentLiterature (PTL) 4). However, this method has a risk such that massadministration may cause the development of a pulmonary embolism, andthus has a safety problem.

As a method for alleviating these problems and providing DDS effects ina disease site-specific manner, the production of a nanosphere(hereinafter sometimes abbreviated as NS) formulation containing, forexample, a PGI2 receptor agonist, such as compound (A), has beenconsidered. There are many known methods for producing NS formulations.NS formulations are known as DDS formulations, which are intravenouslyadministered to utilize vascular permeability enhancement action atinflammatory sites, ischemic sites, and/or cancer tissues; and utilizedisease site-specific drug accumulation. However, the production of aclinically applicable NS formulation comprising a PGI2 receptor agonist,such as compound (A), has been difficult due to the stability, content,yield, safety, sustained release rate, efficacy, etc., of theformulation. In addition, it was extremely difficult to produce an NSformulation containing compound (A) capable of accumulation at a diseasesite and exhibiting the effect.

Methods for producing an NS formulation are roughly classified intobreakdown methods and build-up methods. Breakdown methods are methods ofpulverizing particles by spray-drying or a like method to reduce theparticle size to submicron size. Build-up methods are known to produce,for example, polymer capsule formulations comprising a lacticacid-glycolic acid copolymer (PLGA), a lactic acid polymer (PLA), etc.;drug-encapsulating micelle formulations comprising micellarnanoparticles (polymer micelles) that have a two-layer structurecomprising a block copolymer (copolymer) formed by combiningpolyethylene glycol (PEG) and a polyamino acid; hydrogel formulationsproduced by crosslinking gelatin, collagen, or a polymer mixture ofhyaluronic acid, alginic acid, and the like, to form a hydrogel, andimmobilizing a cell growth factor or the like in the hydrogel; andliposomal formulations having a drug encapsulated in variousphospholipids.

When fine particles have a size as small as several nanometers or less,the particles are excreted from the kidney into urine, and cannot beretained in the body. On the other hand, when fine particles have a sizeof 400 nm or more, the fine particles are quickly eliminated from thebody due to the immune mechanism of eliminating foreign matter bymacrophages or the like. Therefore, as an NS formulation containing adrug, an NS formulation of several nanometers to 400 nm is recommendeddue to its enhanced permeation and retention effect (EPR effect). Morespecifically, unlike normal vascular endothelial cells, there is a widegap of about 200 nm between vascular endothelial cells in cancer tissueor at an inflammation or ischemic site; it is known that a microparticleformulation having a size controlled to about 100 nm, or a polymerformulation, can be accumulated in tissue of vascular lesions created bycancer, infectious disease, ischemia, inflammation, arteriosclerosis,rheumatism, or the like. Thus, as an NS formulation having a DDS effect,there is known a method of forming an NS formulation having a particlesize adjusted to 50 nm to 200 nm to allow a drug to reach a lesion siteand release the drug at the lesion site, thus enhancing its therapeuticeffect. Further, as an endocytosis effect, NS formulations are known topass through a cell membrane and exhibit effects in cells. It is knownto produce, for example, an oral nanosphere formulation havingcalcitonin encapsulated in lactic acid-glycolic acid copolymer (PLGA)nanoparticles (Non-patent Literature (NPL 1)); a transpulmonarynanosphere formulation having calcitonin encapsulated in chitosannanoparticles (NPL 2); a topical nanosphere formulation having steroidencapsulated in PLGA nanoparticles (NPL 3); and a nanosphere formulationhaving an anti-inflammatory agent or a mitochondrial injury inhibitorencapsulated in lactic acid-glycolic acid copolymer (PLGA)nanoparticles. Such nanosphere formulations are effective for treatingischemic reperfusion injury (Patent Literature (PTL) 5). Further, ananosphere formulation having prostaglandin E1 or a derivative thereofencapsulated in lactic acid-glycolic acid copolymer (PLGA) nanoparticlesis also known (PTL 6 and NPL 4). Further, a nanosphere formulationcomprising beraprost encapsulated in lactic acid-glycolic acid copolymer(PLGA) nanoparticles is known to be effective for pulmonary hypertension(PTL 7).

Since an encapsulated drug is released from such a PLGA or PLAnanoparticle formulation by hydrolysis of a lactic acid-glycolic acidbond with water, a nanoparticle formulation having a large surface areahas a very short drug-release time. In contrast, the liposomalformulation gradually releases the drug through enzymatic degradation bylipase etc. in vivo.

A pharmaceutical composition comprising, as an active ingredient, aliposome in which an immunosuppressive agent, such as FK506, FTY720, orcyclosporin A, is encapsulated is also known to be effective fortreating cardiovascular inflammatory diseases, such as myocardialinfarction, myocarditis, and vasculitis syndrome (PTL 8). Doxil(produced by Janssen Pharmaceutical K.K.) comprising doxorubicin (ananticancer antibiotic) encapsulated in liposomes has already beencommercially available as an anticancer drug. This pharmaceuticalcomposition is also commercially available for other purposes, such asan antifungal agent, a Kaposi's sarcoma inhibitor, a lymphomatousmeningitis inhibitor, an age-related macular degeneration inhibitor, anda postoperative pain inhibitor. LipoPGE₁, which is encapsulated in lipidmicrospheres in the form of an o/w emulsion of prostaglandin E1comprising egg yolk lecithin, oleic acid, olive oil, and glycerin, isalready commercially available (Ripple, sold by Mitsubishi Tanabe PharmaCorporation) (NPL 4). LipoPGE₁, which has an average particle size aslarge as 200 to 300 nm and has no stealth property, is trapped by theliver and macrophages, and thus has a short blood retention time.

On the other hand, no stealth liposomal formulation comprising aprostaglandin I2 receptor agonist and having an average particle size of50 to 200 nm has been reported.

CITATION LIST Patent Literature

-   PTL 1: JPH6-87811A-   PTL 2: WO2004/032965-   PTL 3: WO2008/047863-   PTL 4: WO2014/069401-   PTL 5: WO2016/006577-   PTL 6: WO2010/058669-   PTL 7: JP2012-171883A-   PTL 8: WO2013/176223

Non-Patent Literature

-   NPL 1: Y. Kawashima, H. Yamamoto, H. Takeuchi and Y. Kuno, Pharm.    Develop. Technol., 5, 77-85, (2000)-   NPL 2: Y. Kawashima, 6th US-Japan Symposium on Drug Delivery    Systems, December 16-21, (2001), Maui-   NPL 3: E. Horisawa et al., Pharm. Res., 19, 132-139, (2002)-   NPL 4: J Pharm Pharmacol., 2013 August; 65(8): 1187-94

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a disease site-specificstealth liposomal formulation that is effective for treating acardiovascular disease, such as ischemic and dilated cardiomyopathy,obstructive arteriosclerosis, vasculitis syndrome, valvular disease,aortic stenosis, chronic heart failure, or diastolic dysfunction; arespiratory disease, such as pulmonary hypertension, pulmonary fibrosis,asthma, or chronic obstructive pulmonary disease; a gastrointestinal orurinary disease, such as chronic kidney disease, chronic hepatitis, orchronic pancreatitis; and a neurodegenerative disease, such as cerebralinfarction chronic stage, Alzheimer's disease, diabetic neuropathy,Parkinson's disease, or amyotrophic lateral sclerosis.

More specifically, an object of the present invention is to provide aclinically applicable, safe and convenient, stealth liposomalformulation, which is a liposome (LP) formulation containing, forexample, a PGI2 receptor agonist compound (A), and which isintermittently administered by intravenous injection, inhalation, or thelike; and thereby specifically accumulated at a disease site, thusexhibiting a DDS effect.

Solution to Problem

The present inventors conducted extensive research on the production ofNS formulations having a PGI2 receptor (IP) agonist encapsulatedtherein. As a result, the inventors found that a stealth liposome(hereinafter sometimes abbreviated as LP) formulation is the optimumformulation to achieve this object. Through the research of manyproduction methods, the inventors found an LP formulation that isclinically applicable in terms of stability, content percentage, yield,safety, efficacy, release, stealth properties, and the like, from amongLP formulations containing compound (A) or the like. More specifically,the inventors found that a liposomal formulation that is a microparticledrug carrier coated with, for example, PEG-modified phosphoethanolamineand phospholipids can improve drug release control and stability, aswell as exhibit new functions, such as accumulation at a disease site(targeting) and adhesion to tissue; thus significantly improvingbioavailability (BA) and drug efficacy and thereby providing anincreased effect at a lower dose than each component used alone, andreducing side effects.

The present inventors conducted intensive research to solve the aboveproblems, and found for the first time that in a liposomal formulationcontaining a PGI2 receptor (IP) agonist, such as compound (A), anappropriate combination of the types and composition ratio of lipidssuch as a phospholipid component and PEG-modified phosphoethanolaminehaving stealth properties; the average particle size of the liposomalformulation; the weight ratio of compound (A) or the like to thephospholipid; etc., surprisingly allows for the control of the releaserate of a PGI2 receptor agonist, such as compound (A), which is a lowmolecular compound, and thus improves accumulation at a disease site,thereby exhibiting a DDS effect.

The present inventors further found that a specific combination oflipids allows the liposomal formulation to retain stealth properties, sothat liposomes can escape capture by macrophages or the like. Further,the inventors found that the method of the present invention canreliably produce a stealth liposomal formulation with a high yield. Thepresent invention has been accomplished through further trial and errorbased on these findings, and includes the following inventions.

Item 1

A pharmaceutical composition for disease site-specific treatment,comprising a stealth liposome having a prostaglandin I2 receptor agonistencapsulated therein.

Item 2

The pharmaceutical composition according to Item 1, wherein theprostaglandin I2 receptor agonist includes at least a compoundrepresented by formula (I):

wherein

(wherein e represents an integer of 3 to 5,

f represents an integer of 1 to 3,

p represents an integer of 1 to 4,

q represents 1 or 2, and

r represents an integer of 1 to 3);

R¹ represents a hydrogen atom or a C₁₋₄ alkyl group;

R² represents (i) a hydrogen atom, (ii) a C₁₋₈ alkyl group, (iii) aphenyl group or a C₄₋₇ cycloalkyl group, (iv) a 4- to 7-memberedmonocyclic ring containing one nitrogen atom, (v) a C₁₋₄ alkyl groupsubstituted with a benzene ring or a C₄₋₇ cycloalkyl group, or (vi) aC₁₋₄ alkyl group substituted with a 4- to 7-membered monocyclic ringcontaining one nitrogen atom; and

R³ represents (i) a C₁₋₈ alkyl group, (ii) a phenyl group or a C₄₋₇cycloalkyl group, (iii) a 4- to 7-membered monocyclic ring containingone nitrogen atom, (iv) a C₁₋₄ alkyl group substituted with a benzenering or a C₄₋₇ cycloalkyl group, or (v) a C₁₋₄ alkyl group substitutedwith a 4- to 7-membered monocyclic ring containing one nitrogen atom;

(provided that when

is a group represented by (iii) or (iv), —(C—(CH₂)_(p)— and—CH—(CH₂)_(s)— are bound to position a or b on the ring, and cyclicstructures in R² and R³ are optionally substituted with one to threeC₁₋₄ alkyl groups, C₁₋₄ alkoxy groups, halogen atoms, nitro groups, ortrihalomethyl groups); or

a salt thereof

Item 3

The pharmaceutical composition according to Item 1, wherein theprostaglandin I2 receptor agonist includes at least the followingcompound (A):

(A)({5-[2-({[(1E)-phenyl(pyridin-3-yl)methylene]amino}oxy)ethyl]-7,8-dihydronaphthalen-1-yl}oxy)aceticacid (ONO-1301) represented by formula (II):

or a salt of compound (A).

Item 4

The pharmaceutical composition according to Item 1, wherein theprostaglandin I2 receptor agonist includes at least one of the followingcompounds (B) to (E):

(B) sodium(±)-(1R,2R,3aS,8bS)-2,3,3a,8b-tetrahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-hydroxy-4-methyl-1-octen-6-ynyl]-1H-cyclopenta[b]benzofran-5-butanoate(beraprost); or a derivative thereof that is a carbacyclic PGI2derivative,

(C) MRE-269,

(D)(2E)-7-{(1R,2R,3R)-3-hydroxy-2-[(1E,3S,5S)-3-hydroxy-5-methylnon-1-en-1-yl]-5-oxycyclopentyl}hept-2-enoicacid (limaprost), omoprostil, enprostil, or misoprostol; or a derivativeof any of these compounds that is a PEF derivative, and

(E) NS-304 (selexipag);

or

a salt of any of compounds (B) to (E).

Item 5

The pharmaceutical composition according to Item 1, wherein theprostaglandin I2 receptor agonist includes at least one member selectedfrom the group consisting of ONO-1301, beraprost, limaprost, and NS-304.

Item 6

The pharmaceutical composition according to any one of Items 1 to 5,wherein the stealth liposome is obtainable by using at least aprostaglandin I2 receptor agonist and a phospholipid by the Banghammethod, hydration dispersion method, reverse phase evaporation method,ethanol injection method, ethanol dilution method, homogenizationmethod, mechanochemical method, direct dispersion method, extrudermethod, French press method, remote loading method,dehydration-rehydration method, freeze-thaw method, ultrasonic method,or lipid-compound film method; or a modified method of any of thesemethods.

Item 7

The pharmaceutical composition according to any one of Items 1 to 6,wherein the stealth liposome has an average particle size of 50 to 200nm, and comprises 5 to 50 parts by weight of the phospholipid and 0.05to 5 parts by weight of PEG-modified phosphoethanolamine, per part byweight of the prostaglandin I2 receptor agonist.

Item 8

The pharmaceutical composition according to any one of Items 1 to 7,wherein the stealth liposome comprises 0.05 to 5 parts by weight ofMPEG2000-DSPE per part by weight of the prostaglandin I2 receptoragonist; the prostaglandin I2 receptor agonist includes at least onemember selected from the group consisting of ONO-1301, beraprost,limaprost, and NS-304; and the stealth liposome releases theprostaglandin I2 receptor agonist over a period of 3 hours to 4 weeks.

Item 9

The pharmaceutical composition according to any one of Items 1 to 8,wherein the stealth liposome comprises

a prostaglandin I2 receptor agonist,

a phospholipid,

a PEG-modified phosphoethanolamine, and

a water-miscible organic solvent, and

does not comprise a sterol;

the liposome is obtainable by a production method comprising thefollowing steps (1) to (8):

(1) mixing the prostaglandin I2 receptor agonist, the phospholipid, andthe PEG-modified phosphoethanolamine in the solvent in amounts such thatat least 5 mg of the phospholipid and at least one 0.05 mg of thePEG-modified phosphoethanolamine are present per mg of the prostaglandinI2 receptor agonist,

(2) heating the mixture obtained in step (1) to prepare a melt,

(3) instantly freezing the melt obtained in step (2),

(4) freeze-drying the frozen product obtained in step (3) to remove thesolvent,

(5) heating the freeze-dried product obtained in step (4) to dispersethe heated product in an aqueous phosphate buffer solution,

(6) sizing the dispersion obtained in step (5) with an extruder,

(7) ultrafiltrating the dispersion obtained in step (6) to removeunencapsulated material, and

(8) adding a sugar to the dispersion obtained in step (7) andfreeze-dying the dispersion; and the liposome contains at least 0.001 mgof the prostaglandin I2 receptor agonist per 1.0 mg of the phospholipidand has an average particle size of 50 to 200 nm.

Item 10

The pharmaceutical composition according to any one of Items 1 to 9,wherein the composition is for intravenous administration, intracoronaryadministration, inhalation, intramuscular injection, subcutaneousadministration, oral administration, transmucosal administration,transdermal administration, or an internal organ; and is in the form ofan injectable formulation, an oral preparation, an inhalant, anebulizer, an ointment, a patch, or a spray.

Item 11

The pharmaceutical composition according to any one of Items 1 to 9,wherein a single intravenous dose of the composition is 0.001 to 100 mgin terms of the prostaglandin I2 receptor agonist.

Item 12

The pharmaceutical composition according to Item 11, wherein a diseaseto be treated with the composition is cardiovascular disease,respiratory disease, urinary disease, gastrointestinal disease, bonedisease, neurodegenerative disease, vascular disease, dental disease,eye disease, skin disease, other inflammatory disease, ischemic organdisorder, diabetic complication, tissue fibrotic disease, tissuedegenerative disease, or hair loss; and

the composition comprises a liposome.

Item 13

The pharmaceutical composition according to Item 11, wherein the diseaseto be treated with the composition is a cardiovascular disease such asischemic and dilated cardiomyopathy, atherosclerosis obliterans,vasculitis syndrome, valvular disease, aortic stenosis, chronic heartfailure, or diastolic failure; a respiratory lung disease such aspulmonary hypertension, pulmonary fibrosis, asthma, or chronicobstructive pulmonary disease; a gastrointestinal or urinary diseasesuch as chronic kidney disease, chronic hepatitis, or chronicpancreatitis; or a neurodegenerative disease such as chronic phase ofcerebral infarction, Alzheimer's disease, diabetic neuropathy,Parkinson's disease, or amyotrophic lateral sclerosis; and

the composition comprises a liposome.

Advantageous Effects of Invention

According to the present invention, there can be provided apharmaceutical composition that is effective for, for example,cardiovascular diseases, respiratory diseases, gastrointestinal orurinary diseases, and inflammatory diseases such as neurodegenerativediseases, ischemic organ disorders, diabetic complications, tissuefibrotic diseases, tissue degenerative disease, or hair loss. Thepharmaceutical composition of the present invention can have the effectof enhancing drug efficacy at a lower dose than a single use of a PGI2receptor agonist, and also reducing side effects. Further, the presentinvention can provide a stealth liposomal formulation that allows foraccumulation of a PGI2 receptor agonist in a high concentration at adisease site, and exhibit effects in a sustained manner; and provide amethod for producing the stealth liposomal formulation.

The liposomal formulation of the present invention containing compound(A) or the like is effective for circulatory diseases, respiratorydiseases, urinary diseases, gastrointestinal diseases, andneurodegenerative diseases, when intermittently administered byintravenous administration, intramuscular administration, subcutaneousadministration, or inhalation administration. In particular, theliposomal formulation administered intravenously or by inhalation isaccumulated at a disease site and topically exhibits an endogenousrepair factor production-promoting action, thus being useful as aregenerative drug. Intravenous administration of the liposomalformulation is useful for heart diseases, such as myocardial infarction,angina, dilated cardiomyopathy, aortic stenosis, valvular disease,chronic heart failure, and diastolic dysfunction, due to its vasodilatoraction, angiogenesis action, stem cell differentiation-inducing action,antifibrotic action, antiapoptotic action, reverse remodeling action,etc. For pulmonary diseases or the like such as acute pneumonia, chronicpneumonia, pulmonary hypertension, pulmonary fibrosis, interstitialpneumonia, COPD, and ARDS, inhalation administration, in addition tointravenous administration, is useful. For neurodegenerative diseasessuch as amyotrophic lateral sclerosis (ALS), Parkinson's disease,Alzheimer's disease, and spinal cord injury, intramedullaryadministration, in addition to intravenous administration, is useful.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is diagrams showing the average particle size distribution ofFormulations 1 to 4.

FIG. 2 is diagrams showing the average particle size distribution ofFormulations 5 to 8.

FIG. 3 is a diagram showing the average particle size distribution ofFormulation 9.

FIG. 4 is a diagram showing the average particle size distribution ofFormulation 10.

FIG. 5 is diagrams showing the average particle size distribution ofFormulations 11 to 14.

FIG. 6 is diagrams showing the average particle size distribution ofFormulations 15 to 18.

FIG. 7 is a diagram showing the average particle size distribution ofFormulation 19.

FIG. 8 is a diagram showing the average particle size distribution ofFormulation 20.

FIG. 9 is a graph showing the results of quantification of compound (A).

FIG. 10 is a diagram showing the average particle size distribution ofFormulation 21.

FIG. 11 is transmission electron microscope images of Formulation 21.

FIG. 12 is charts of HPLC measurement of Samples 1 to 5.

FIG. 13 is a diagram showing the average particle size distribution ofFormulation 22.

FIG. 14 is transmission electron microscope images of Formulation 22.

FIG. 15 is an absorption spectrum of a solution of compound (B).

FIG. 16 is a diagram showing the average particle size distribution ofFormulation 23.

FIG. 17 is transmission electron microscope images of Formulation 23.

FIG. 18 is an absorption spectrum of compound (C).

FIG. 19 is diagrams showing the average particle size distribution ofFormulations 24 and 25.

FIG. 20 shows the results of HPLC analysis.

FIG. 21 shows the particle size distribution of liposomes havingcompound (B) encapsulated therein (Formulation 26).

FIG. 22 shows UV absorption spectra of compound (B), and liposomeshaving compound (B) encapsulated therein.

FIG. 23 shows the particle size distribution of liposomes havingcompound (D) encapsulated therein (Formulation 27).

FIG. 24 is UV absorption spectra of compound (D), and liposomes havingcompound (D) encapsulated therein (Formulation 27).

FIG. 25 is a diagram showing the average particle size distribution ofliposomes having compound (E) encapsulated therein (Formulation 28).

FIG. 26 is absorption spectra of compound (E) and liposomes havingcompound (E) encapsulated therein (Formulation 28).

FIG. 27 shows 42-day survival curves of a group receiving ONO-1301 byrepeated oral administration, and a group receiving Formulation 21(ONO-1301LipoNS formulation) by intermittent intravenous administration.

FIG. 28 is a graph showing the survival rates of all groups.

FIG. 29 is a graph showing a comparison with intermittent intravenousadministration of Formulation 25 (ONO-1301Lipo).

FIG. 30 is a graph showing a comparison with intermittent intratrachealadministration of Formulation 25 (ONO-1301Lipo).

FIG. 31 is a drawing showing a method for evaluating a left ventriclewall thickness and a left ventricle wall area.

FIG. 32 is photographs showing an infarct area evaluation method.

DESCRIPTION OF EMBODIMENTS

The liposomes used in the pharmaceutical composition of the presentinvention are not limited, as long as they are closed vesiclessurrounded by a lipid bilayer. The liposomes may be large unilamellarvesicle (LUV) liposomes, or small unilamellar vesicle (SUV) liposomes;and may be multilamellar vesicle (MLV) liposomes. The liposomes can beproduced by known production methods.

There are three types of methods for producing liposomes. Morespecifically, the Bangham method is commonly used as a liposomeproduction method. There are also methods comprising the Bangham methodand some additional operations, which are called the simple hydrationmethod, ultrasonic treatment method, and extrusion method. Examples ofliposome production methods further include the direct dispersionmethod, organic solvent (e.g., ethanol) injection method, reverse phaseevaporation method, calcium fusion method, surfactant removal method,static hydration method, hexane-span 80 dialysis method, organic solventglobule evaporation method, mechanochemical method, ultrasonic method,lipid-compound film method, and the like; and improved methods of thesemethods.

The method for adjusting the particle size includes the extrusionmethod, extrusion process, French press method, and the like. Examplesof the extruder method or the French press method includes a methodcomprising passing particles several times through a nanopore membranefilter having an appropriate pore size, which is set in an extruder or aFrench press to adjust the liposome size.

Examples of the method for encapsulating the compound include the pHgradient (remote loading) method, counter ion concentration gradient(gelation) method, freeze-thaw method, supercritical carbon dioxidemethod, film loading method, and the like.

The methods that are superior in encapsulating a water-soluble druginclude the reverse phase evaporation method and the freeze-thaw method.The methods that are superior in encapsulating fat-soluble drugs includethe Bangham method, the mechanochemical method, the supercritical carbondioxide method, and the film loading method. The methods that aresuperior in encapsulating dissociative drugs include the pH gradient(remote loading) method, the counterionization concentration gradientmethod, and the like.

The Bangham method includes, for example, a method comprising forming alipid film; and then applying a mechanical vibration by vortexing,ultrasonic waves, or the like in an aqueous buffer to form liposomes (ahydration dispersion method). The reverse phase evaporation methodincludes, for example, a method comprising dissolving a lipid in anorganic solvent that is immiscible with water; then adding an aqueousbuffer and performing ultrasonic treatment to form a reverse micelle (aW/O emulsion), thereafter removing the organic solvent by vacuumtreatment or the like and achieving a gel state; and then formingliposomes. The ethanol injection method or the ethanol dilution methodincludes, for example, a method comprising dissolving a lipid inethanol, and injecting a lipid solution into an aqueous buffer to formliposomes.

The homogenization method or the mechanochemical method includes, forexample, a method of forming liposomes by using a high-pressureemulsifier.

The direct dispersion method includes a method comprising directlydispersing a lipid or a mixture of a lipid and a compound in an aqueousbuffer, without preparing a lipid film, to form liposomes.

Among the methods for adjusting the particle size, the extruder methodor the French press method includes, for example, a method comprisingpassing the liposomes through a nanopore membrane set in an extruder ora French press to thereby adjust the liposome size.

Among the methods of encapsulating the compound, the remote loadingmethod is an encapsulation method utilizing the difference in pHsolubility of the compound. More specifically, after liposomes having asan inner aqueous phase a pH solution in which the compound iswater-soluble are formed, the outer aqueous phase is replaced with a pHsolution in which the compound is fat-soluble by ultrafiltration,dialysis, or the like; and adding a compound solution to the liposomedispersion to thereby encapsulate the compound in the aqueous phase ofthe liposomes. The dehydration-rehydration method is an encapsulationmethod in which liposomes are dehydrated by freeze-drying or the like;and then rehydrated with an aqueous buffer containing a compound to beencapsulated, thereby encapsulating the compound.

The freeze-thawing method includes, for example, a method comprisingmixing a liposome dispersion and a compound solution to be encapsulated,and repeating freeze-thaw cycles to thereby encapsulate a compound at ahigh concentration.

The method for producing liposomes is not limited to the productionmethods described above. The method further includes improved methods ofeach of these methods, and the like.

The lipids that form liposomes are not particularly limited. Examples oflipids include soy lecithin, hydrogenated soy lecithin, egg yolklecithin, phosphatidylcholines, phosphatidylserines,phosphatidylethanolamines, phosphatidylinositols, phosphasphingomyelins,phosphatidic acids, long-chain alkyl phosphates, gangliosides,glycolipids, phosphatidylglycerols, sterols, and the like. Lipids can beused singly, or in a combination of two or more. Examples ofphosphatidylcholines include dimyristoylphosphatidylcholine,dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, and thelike. Examples of phosphatidylserines include dipalmitoylphosphatidylserine, sodium dipalmitoylphosphatidylserine, bovinebrain-derived sodium phosphatidylserine, and the like. Examples ofphosphatidylethanolamines include dimyristoyl phosphatidylethanolamine,dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine,and the like. Examples of the phosphatidylinositols includewheat-derived phosphatidylinositol sodium, and the like. Examples ofphosphasphingomyelins include bovine-derived sphingomyelin, and thelike. Examples of phosphatidic acids and long-chain alkyl phosphatesinclude dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid,distearoyl phosphatidic acid, dicetyl phosphoric acid, and the like.Examples of gangliosides include ganglioside GM1, ganglioside GD1a,ganglioside GT1b, and the like. Examples of glycolipids includegalactosylceramide, glucosylceramide, lactosylceramide, phosphatide,globoside, and the like. Examples of phosphatidyl glycerol includedimyristoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol,distearoyl phosphatidyl glycerol, and the like. Examples of sterolsinclude cholesterol, dihydrocholesterol, lanosterol, dihydrolanosterol,sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, andthe like. When two or more lipids are used in combination, aphospholipid and cholesterol are preferably used in combination. Thephospholipid is preferably a phosphatidylcholine. When liposomes areproduced by using a phospholipid and cholesterol, the molar ratio of thephospholipid to cholesterol is preferably in the range of 1:0.1 to1:1.5, and more preferably 1:0.5 to 1:1.25.

Examples of phospholipids that can be used in the present inventioninclude the following commercially available products (sold by NipponFine Chemical Co., Ltd.).

In general, the phospholipid is preferably DOPC or DEPC, although it mayvary depending on the substance to be encapsulated therein.

TABLE 1 Abbreviated product name IUPAC name Cas Reg. No. DPPC1,2-Dipalmitoyl-sn- 63-89-8 glycero-3-phosphocholine DSPC1,2-Distoaroyl-sn- 816-94-4 glycero-3-phosphocholine DMPC 1,2-Dimy

is

oyl- 18194-24-6 sn-glycerol-3-phosphocholine DOPC1,2-Dioleoyl-sn-glycero- 4235-96-4 3-phosphocholine DBPC1,2-Dieracoyl-sn-Glycero- 51779-95-4 3-Phosphocholine POPC2-Oleoyl-1-palmitoyl-sn- 26853-31-6 glycero-3-phosphocholine PCS1,2-Diacyl-sn-Glycero- 8002-43-5 3-Phosphocholine (SOY) PCSH1,2-Diacyl-sn-Glycero- 8002-43-5 3-Phosphocholine (SOY) DPPG1,2-Dipalmitoyl-sn-Glycero-

- 67233-81-9 [Phospho-rac-(1-glycerol)] (Sodium Salt) DMPG 1,2-Dimy

istoyl-sn-Glycero-3- 67232-80-8 [Phospho-rac-(1-glycerol)] (Sodium Salt)DSPG 1,2-Dist

aroyl-sn-Glycero-3- 4537-78-4 [Phospho-rac-(1-glycerol)] (Sodium Salt)DOPG 1,2-Diole

yl-sn-Glycero-3- 62706-09-0 [Phospho-rac-(1-glycerol)] (Sodium Salt) PGE1,2-Diacyl-sn-Glycero-3- N/A [Phospho-rac-(1-glycerol)] (Sodium Salt,EGG) PGS 1,2-Diacyl-sn-Glycero-3- N/A {Phospho-rac-(1-glycerol)] (SodiumSalt, SOY) PGSH 1,2-Diacyl-sn-Glycero-3- N/A [Phospho-rac-(1-glycerol)](Sodium Salt, SOY)

indicates data missing or illegible when filed

Conventional liposomal formulations are liposomes comprising typicalphospholipid and cholesterol. There are also stealth liposomes whosesurface is modified with polyethylene glycol (PEG) or the like toincrease the blood retention. These liposomes have the effect ofaccumulation specifically at a disease site due to their EPR effects.

To increase the stability (stealth properties) of liposomes in blood,the liposome membrane surface is preferably modified with a polyethyleneglycol (PEG) derivative. The liposomes modified with a PEG derivativecan be produced by using a covalent conjugate of PEG having a molecularweight of 500 to 20000 and a phospholipid. The covalent conjugate of PEGand phospholipid is preferably a PEG-modified phosphoethanolamine, whichis a conjugate of PEG having a molecular weight of 200 to 5000 anddistearoylphosphatidylethanolamine.

Examples of PEG-modified phosphoethanolamines include commerciallyavailable products, such as DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DSPE(1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and the like (allproduced by Nippon Fine Chemical Co., Ltd.), which comprise mPEG 350,mPEG 550, mPEG 750, mPEG 1000, mPEG 2000, mPEG 3000, or mPEG 5000 as aPEG-modifying group.

A preferable combination is, for example, a combination ofmPEG2000-DSPE: N-(carbonyl-methoxypolyethyleneglycol 5000)-1,2distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt CAS No.147867-65-0 (produced by Nippon Fine Chemical Co., Ltd.) and DEPC:1,2-dierucoyl-sn-glycerol-3-phosphorylcholine CAS No. 51779-95-4(produced by Nippon Fine Chemical Co., Ltd.).

The contents of the PEG-modified phosphoethanolamine and phospholipidare not particularly limited. The content of the PEG-modifiedphosphoethanolamine is preferably 0.01% to 10%, and more preferably0.01% to 3%, relative to the phospholipid as 1.

There is also a method in which the liposome surface is modified withPEG and a targeting molecule, such as an antibody or a peptide, toincrease blood retention and further enhance the transfer to a targetsite.

The present invention provides a stealth liposome characterized in thatthe liposome contains a PGI2 receptor agonist and a phospholipid, andfurther comprises PEG-modified phosphoethanolamine. The liposome ispreferably formed into a liposomal formulation by combining a PGI2receptor agonist and a phospholipid, and further EG-modifiedphosphoethanolamine, according to the purpose; and mixing thesecomponents at an appropriate ratio.

The stealth liposome comprises a prostaglandin I2 receptor agonist, aphospholipid, a PEG-modified phosphoethanolamine, and a water-misciblesolvent; and can be produced, for example, by a production methodcomprising the following steps:

mixing a prostaglandin I2 receptor agonist, a phospholipid, and aPEG-modified phosphoethanolamine in a water-miscible solvent in amountssuch that at least 5 mg of the phospholipid and at least 0.05 mg of thePEG-modified phosphoethanolamine are present per mg of the prostaglandinI2 receptor agonist, to prepare a mixture;

heating the mixture to prepare a melt;

instantly freezing the melt;

freeze-drying the frozen product to remove the solvent;

heating the freeze-dried product to disperse the heated product in anaqueous phosphate buffer solution;

sizing the dispersion with an extruder;

ultrafiltrating the dispersion to remove unencapsulated material; and

adding a sugar to the dispersion, and freeze-drying the dispersion.

The liposomes produced by this method are a stealth liposomalformulation characterized by containing a PGI2 receptor agonist, andhaving an average particle size of 50 to 200 nm.

The mixture of the PGI2 receptor agonist, the phospholipid, PEG-modifiedphosphoethanolamine, and solvent may or may not contain a sterol, suchas cholesterol. Conventional liposomes preferably comprise a combinationof a phospholipid and cholesterol as constituent lipids. However, thepresent invention uses no cholesterol as a constituent lipid, andthereby makes it possible to produce liposomes in which a PGI2 receptoragonist is stably encapsulated at a high concentration.

The size (particle size) of the liposomes is not particularly limited.The liposomes preferably have an average particle size of about 10 to1000 nm, more preferably about 20 to 500 nm, and even more preferablyabout 50 to 200 nm. The “particle size” referred to herein means thediameter of a particle determined by the dynamic light scatteringmethod. A preferable polydispersity index (PDI) is 0.3 or less. Themethod for adjusting the particle size is not particularly limited.

The present invention provides a prophylactic and/or therapeutic agentfor a cardiovascular disease, a respiratory disease, a urinary disease,a vascular disease, a gastrointestinal disease, a neurodegenerativedisease, etc., which comprises, as an active ingredient, liposomeshaving a PGI2 receptor agonist encapsulated therein. The PGI2 receptoragonist used in the pharmaceutical composition of the present inventionis not particularly limited; a known PGI2 receptor agonist can bepreferably used. Examples of known PGI2 receptor agonists include, forexample, pharmaceutical compositions, PGI2 derivatives, and PGEderivatives, which are compounds represented by the following formula(I):

wherein

(wherein e represents an integer of 3 to 5,

f represents an integer of 1 to 3,

p represents an integer of 1 to 4,

q represents 1 or 2, and

r represents an integer of 1 to 3);

R¹ represents a hydrogen atom or a C₁₋₄ alkyl group;

R² represents (i) a hydrogen atom, (ii) a C₁₋₈ alkyl group, (iii) aphenyl group or a C₄₋₇ cycloalkyl group, (iv) a 4- to 7-memberedmonocyclic ring containing one nitrogen atom, (v) a C₁₋₄ alkyl groupsubstituted with a benzene ring or a C₄₋₇ cycloalkyl group, (vi) a C₁₋₄alkyl group substituted with a 4- to 7-membered monocyclic ringcontaining one nitrogen atom; and

R³ represents (i) a C₁₋₈ alkyl group, (ii) a phenyl group or a C₄₋₇cycloalkyl group, (iii) a 4- to 7-membered monocyclic ring containingone nitrogen atom, (iv) a C₁₋₄ alkyl group substituted with a benzenering or a C₄₋₇ cycloalkyl group, (v) a C₁₋₄ alkyl group substituted witha 4- to 7-membered monocyclic ring containing one nitrogen atom;

(provided that when

is a group represented by (iii) or (iv),

—(C—(CH₂)_(p)— and ═CH—(CH₂)_(s)— are bound to position a or b on thering, and cyclic structures in R² and R³ are optionally substituted withone to three C₁₋₄ alkyl groups, C₁₋₄ alkoxy groups, halogen atoms, nitrogroups, or trihalomethyl groups); and salts thereof.

Preferably, the PGI2 receptor agonist is one of the following compounds:

(A)({5-[2-({[(1E)-phenyl(pyridin-3-yl)methylene]amino}oxy)ethyl]-7,8-dihydronaphthalen-1-yl}oxy)aceticacid (CAS 17639141-6; compound (A)(ONO-1301)) represented by formula(II):

(B) carbacyclic PGI2 derivatives such as sodium(±)-(1R,2R,3aS,8bS)-2,3,3a,8b-tetrahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-hydroxy-4-methyl-1-octene-6-ynyl]-1H-cyclopenta[b]benzofuran-5-butanoate(CAS: 88475-69-8; beraprost)(compound (B));

(C) [4-(5,6-diphenylpyrazinyl)(1-methylethyl)amino]butoxy]-acetic acid(CAS: 475085-57-5; MRE-269; compound (C));

(D) PGE derivatives such as(2E)-7{-(1R,2R,3R)-3-hydroxy-2[-(1E,3S,5S)-3-hydroxy-5-methylnon-1-en-1-yl]-5-oxocyclopentyl}-hept-2-enoicacid (CAS: 74397-12-9; limaprost), omoprostil;175,20-dimethyl-6-oxo-PGE₁ methyl ester, emprostil, and misoprostol(compound (D)); and

(E)2-{4-[(5,6-diphenylpyrazin-2-)yl)(propan-2-yl)amino]butoxy}-N-(methanesulfonyl)acetamide(CAS: 475086-01-2; selexipag; NS-304 (compound (E)).

The subject to which the pharmaceutical composition of the presentinvention is administered is preferably a mammal having an inflammatorydisease, ischemic organ disorder, diabetic complication, tissue fibroticdisease, tissue degenerative disease, or the like. Examples of mammalsinclude humans, monkeys, cows, sheep, goats, horses, pigs, rabbits,dogs, cats, rats, mice, guinea pigs, and the like. Humans that havedeveloped an inflammatory disease, or humans suspected to have aninflammatory disease, are particularly preferable.

The method for administering the pharmaceutical composition of thepresent invention is not particularly limited, as long as the activeingredient can reach a disease site. Examples include injectableformulations, patches, inhalants, nebulizers, sprays, gels, creams,sprays, ointments, nasal drops, eye drops, and the like, which are forintravenous administration, intracoronary administration, drip/infusion,intracoronary administration, inhalation, intramuscular administration,subcutaneous administration, oral administration, suppositories,intraperitoneal administration, transmucosal administration, transdermaladministration, or internal organs. For intraarterial administration,intracoronary administration is preferable. For intravenousadministration, peripheral intravenous administration is preferable.

The injectable formulation may be either an aqueous injectableformulation or an oily injectable formulation. The aqueous injectableformulation can be prepared by a known method. For example, afterliposomes having a drug encapsulated therein are mixed into a solutionprepared by appropriately adding pharmaceutically acceptable additivesto an aqueous solvent (e.g., water for injectable formulation orpurified water), the resulting mixture is filtered through a filter orthe like and sterilized, and the filtrate is filled into an asepticcontainer. Examples of pharmaceutically acceptable additives includeisotonic agents such as sodium chloride, potassium chloride, glycerin,mannitol, sorbitol, boric acid, borax, glucose, and propylene glycol;buffers such as phosphoric acid buffer, acetic acid buffer, boric acidbuffer, carbonic acid buffer, citric acid buffer, Tris buffer, glutamicacid buffer, and epsilon-aminocaproic acid buffer; preservatives such asmethyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate,butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkoniumchloride, sodium dehydroacetate, sodium edetate, boric acid, and borax;thickeners such as hydroxyethyl cellulose, hydroxypropyl cellulose,polyvinyl alcohol, and polyethylene glycol; stabilizers such as sodiumbisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbicacid, and dibutylhydroxytoluene; pH adjusters such as hydrochloric acid,sodium hydroxide, phosphoric acid, and acetic acid; and the like. Theinjectable formulation may further comprise an appropriate solubilizingagent. Examples of the solubilizing agent include alcohols such asethanol; polyalcohols such as propylene glycol and polyethylene glycol;nonionic surfactants such as polysorbate 80, polyoxyethylene (50)hydrogenated castor oil, lysolecithin, and pluronic polyol; and thelike. The injectable preparation may comprise a protein such as bovineserum albumin or keyhole limpet hemocyanin; a polysaccharide such asaminodextran; and the like. When an oily injectable formulation is to beproduced, for example, sesame oil or soybean oil can be used as an oilysolvent; and benzyl benzoate, benzyl alcohol, or the like can be addedas a solubilizing agent. The prepared injectable formulation is usuallyplaced in, for example, an appropriate ampoule or vial. Liquidformulations such as injectable formulations can also be preserved afterremoving water by cryopreservation, lyophilization, or the like.Lyophilized formulations are dissolved again at the time of use byadding distilled water for injectable formulations or the like; and thenused.

The amount of the drug or the like contained in the pharmaceuticalcomposition of the present invention varies depending on the dosageform, administration interval, or administration route. In the case ofthe injectable formulation for intravenous administration, the amountcan be appropriately selected from the range of 0.001 ng/mL to 100mg/mL. The administration period and administration method areappropriately determined according to the disease and the treatmentmethod therefor, in consideration of safety, convenience, lowinvasiveness, patient's burden, compliance, and the like. Anyadministration interval may be used as long as the effect can beexpected and the administration interval is convenient. Theadministration interval is preferably about twice a day, every day, onceevery two days, once every three days, once a week, once every twoweeks, once every three weeks, or once every four weeks; and is morepreferably in the range of once a day to once a week.

For example, when liposomes having compound (A) encapsulated therein areintravenously administered to a human that has developed a heartdisease, a single dose in terms of ONO-1301 is preferably 500 mg orless, and more preferably 100 mg or less. The lower limit is notparticularly limited, and can be any dose that provides the desiredeffect.

When a PGI2 receptor agonist alone is administered at a high dose, thePGI2 receptor exhibits a hypotensive effect due to its vasodilatoiyeffect; therefore, there is little deviation from the effective amount.In contrast, the pharmaceutical composition of the present invention,which comprises, as an active ingredient, liposomes having a PGI2receptor agonist encapsulated therein, is useful in that the liposomalformulation can exhibit effects on many diseases even at a low dose dueto its accumulation at a disease site by the DDS effect. That is,vascular permeability at a lesion site, and nano-sized liposomes can beexpected to specifically accumulate in the lesion site (EPR effect).Furthermore, the liposomal formulation is highly useful in that sincethe drug moves into cells due to the endocytosis effect, an increase indrug efficacy and a reduction in side effects can be expected. Further,the pharmaceutical composition of the present invention is useful inthat an active ingredient can be delivered to a target lesion site byadministration through a peripheral vein or the like, without thenecessity of using a central venous catheter or the like. Anotheradvantage is that the pharmaceutical composition is difficult to bedelivered to sites other than the target site even when administeredthrough a peripheral vein. In other words, the pharmaceuticalcomposition of the present invention is highly useful in that thecomposition can provide an enhanced tissue repair effect at a low dose;and can reduce side effects by reducing the dose, suppressing deliveryto sites other than the target site, and eliminating the necessity ofusing a central venous catheter or the like.

When a liposomal formulation is to be produced by the direct dispersionimprovement method, and when a premix of lipids is produced, the solventused must meet the following conditions: it is a solvent in which lipidsand the substance to be encapsulated are soluble; it can be instantlyfrozen; and it can be removed by freeze-drying. Any solvent thatsatisfies the above conditions can be used.

In general, the solvent is preferably t-butanol, cyclohexane+ethanol,hexafluoropropanol, 1-propanol, isopropyl alcohol, 2-butoxyethanol, andthe like. t-Butanol is more preferable.

In the freeze-drying of liposomes, it is generally necessary to add anddisperse a sugar, such as maltose, sucrose, or trehalose, to therebyinhibit cell membrane collapse on freezing and perform freeze-drying.

Application to Pharmaceutical Products

PGI2 receptor agonists, such as compound (A), have, for example, an invivo regeneration factor production-promoting action, stem celldifferentiation-inducing action, anti-apoptotic action, reverseremodeling action, anti-fibrotic action, and angiogenesis-promotingaction. Therefore, stealth liposomal formulations containing such a PGI2receptor agonist are useful as therapeutic and/or prophylactic agentsfor the following various diseases:

various organ disorders, inflammatory diseases such as vascular diseases(e.g., atherosclerosis obliterans (ASO), Berger disease, Raynaud'sdisease, arteriosclerosis, vasculitis syndrome, etc.), cardiovasculardiseases (e.g., myocardial infarction, myocarditis, angina,supraventricular tachyarrhythmia, congestive heart failure, coronaryartery disease, idiopathic cardiomyopathy, dilated cardiomyopathy,ischemic cardiomyopathy, atrial fibrillation, chronic heart failure,diastolic dysfunction, systolic dysfunction, valvular disease, aorticstenosis, etc.), neurodegenerative diseases (e.g., ischemicencephalopathy, cerebrovascular disease, stroke, Parkinson's disease,Alzheimer's disease, diabetic neuropathy, spinal canal stenosis,dementia, moyamoya disease, spinal cord injury, muscle atrophy lateralsclerosis (ALS) etc.), respiratory diseases (e.g., acute pneumonia,pulmonary fibrosis, pulmonary hypertension, chronic obstructivepulmonary disease (COPD), systemic inflammatory response syndrome(SIRS), acute lung injury (ALI), acute respiratory distress syndrome(ARDS), Sarcoidosis, interstitial pneumonia, irritable pneumonia,asthma, refractory asthma, etc.), bone diseases (e.g., osteoarthritis(OA) of, for example, spine or knee, rheumatoid arthritis (RA),osteoporosis, fracture, spinal cord injury, periosteal injury, etc.),gastrointestinal liver diseases (e.g., fulminant hepatitis, acutehepatitis, cirrhosis, chronic hepatitis, fatty liver, steatohepatitis,gastric ulcer, gastritis, intestinal ulcer, etc.), urinary diseases(e.g., acute renal failure, chronic renal failure, glomerular disease,tubulointerstitial disease, renal vasculopathy, cystic kidney disease,toxic nephropathy, tubule transport abnormality, dialysis patient kidneydisorders, nephropathy, nephrotic syndrome, IgA nephropathy, atypicalhemolytic uremic syndrome, acute progressive nephritis syndrome, renalfibrosis, etc.), gastrointestinal pancreatic diseases (e.g., diabetes,chronic pancreatitis, acute pancreatitis, etc.), gastrointestinaldiseases (e.g., esophagitis, gastritis, gastric ulcer, duodenal ulcer,ulcerative colitis, Crohn's disease, etc.), diabetic complications(e.g., diabetic neuropathy, skin ulcer, diabetic nephropathy, diabeticretinopathy, etc.), vascular endothelial cell damages (e.g., preventionof restenosis after percutaneous transluminal coronary angioplasty(PTCA)), dental diseases (e.g., periodontal diseases, tooth extractionwounds, oral wounds, periodontal tissue disorders, etc.), skin diseases(e.g., pressure ulcers, hair loss, etc.), ophthalmic diseases (e.g.,glaucoma, etc.), organ/cell transplantation (e.g., heart, liver,kidneys, lungs, pancreas, pancreatic islet cells, bone marrow, etc.),chronic transplant rejection, and the like. In particular, the liposomalformulation of the present invention has shown promise as a prophylacticand/or therapeutic agent for heart diseases, lung diseases, kidneydiseases, bone diseases, neurodegenerative diseases, liver diseases,pancreatic diseases, autoimmune diseases, allergic syndromes, andvascular diseases.

As body regeneration factors whose product is induced or promoted by aPGI2 receptor agonist, such as compound (A), for example, the followingfactors are known: a vascular endothelial cell growth factor (VEGF), ahepatocyte growth factor (HGF), various fibroblast growth factors(a/bFGF), transforming growth factor-β (TGF-β), a platelet-derivedgrowth factor (PDGF), Angiopoietin, a hypoxia-inducible factor (HIF), aninsulin-like growth factor (IGF), a bone morphogenetic protein (BMP), aconnective tissue growth factor (CTGF), an epidermal growth factor(EGF), a stromal cell-derived factor (SDF-1), a high-mobility group box1 (HMGB1), and the like; and growth factors of their families etc.Examples of other drugs that produce endogenous repair factors describedabove include other PGI2 receptor agonists, EP2 and EP4 receptoragonists of PGE2 receptors, and mixed receptor agonists thereof, and thelike. To achieve the object of the present invention, the drugsmentioned above may be used in place of compound (A). Examples of thedrug that can be used in place of compound (A) include PGI and PGEderivatives, IP, EP2 and EP4 receptor agonists, and the like. Specificexamples include compound (B), aeroprost, ornoprostil, compound (C),compound (D) (limaprost), compound (E), enprostil, misoprostol,ONO-4232, ONO-8055, and the like.

These drugs and liposomes containing the drugs exhibit effects on thesame diseases as those on which compound (A) has effects.

It is also preferable in the present invention that two or more drugsselected from compound (A) and drugs described above are combinedaccording to the purpose, and formed into liposomes. The drugs may becommercially available, or can be easily produced in accordance with aknown method.

The dosage form of the liposome of the present invention includesinjectable formulations, ointments, patches, oral preparations, sprays,and the like, which are for intravenous administration, coronary arteryadministration, inhalation, intramuscular administration, subcutaneousadministration, oral administration, transmucosal administration, andtransdermal administration, or internal organs. In addition to theabove, other examples include implants, transmucosal agents foradministration through the rectum, uterus, oral cavity, or the like,nasal drops, and intravenous drip injections; or a method for continuousadministration into coronary arteries.

Toxicity

As compared with the PGI2 receptor agonist alone, the liposomalformulation of the present invention is less toxic and fully safe foruse as a medicament. Further, the PGI2 receptor agonist has beenconfirmed not to have carcinogenesis initiation and promotion effects ina long-term carcinogenicity test, a medium-term hepatocarcinogenicitytest, etc., using mice and rats.

EXAMPLES

The present invention is described in detail below with reference toExamples; however, the present invention is not limited to theseExamples.

The following are the reagents used. In the Examples, these reagents arereferred to by the following abbreviations.

(1) HSPC (hydrogenated soybean phospholipid, hydrogenated lecithin),product name: COATSOME NC-21 (NOF corporation), CAS: 921228-87-5,92128-87-5

(2) DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine), CAS:1069-79-0 (Nippon Fine Chemical Co., Ltd.)

(3) DEPC: 1,2-Dierucoyl-sn-glycerol-3-phosphorylcholine, CAS: 51779-95-4(Nippon Fine Chemical Co., Ltd.)

(4) MPEG2000-DSPE: N-(carbonyl-methoxypolyethyleneglycol-5000)-1,2distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt, CAS:147867-65-0 (Nippon Fine Chemical Co., Ltd.)

(5) DOPC: 1,2-Dioleoyl-sn-glycero-3-phosphocholine, CAS: 4235-95-4(Nippon Fine Chemical Co., Ltd.)

(6) Cholesterol: Cholesterol HP (NOF Corporation)

(7) PBS(−): Dulbecco's phosphate buffered saline (without Ca and Mg),filtered and sterilized, tested for mycoplasma and endotoxin (productcode: 14249-95) (Nacalai Tesque Inc.)

The PGI2 receptor agonists to be encapsulated in liposome arecommercially available from the companies listed below, and can bepurchased generally.

(i) Compound (A): (ONO-1301), Sigma-Aldrich, CAS: 176391-41-6

(ii) Compound (B): (Beraprost), Cayman Chemical Company, CAS: 88475-69-8

(iii) Compound (C): (MRE-269), Cayman Chemical Company, CAS: 475085-57-5

(iv) Compound (D): (Limaprost), Cayman Chemical Company, CAS: 74397-12-9

(v) Compound (E): (NS-304), Cayman Chemical Company, CAS: 475086-01-2

Below, examples of the production of liposomes of PGI2 receptor agonistsare specifically described in detail.

1. Formulation Example 1 (Remote Loading Method) Preparation of Liposome

1) HSPC (107.4 mg), DSPE (9.0 mg), and cholesterol (35.3 mg) wereweighed and placed in an eggplant flask, and a chloroform/methanolsolution (1/1, v/v) was added and dissolved so that the lipidconcentration was 20 mg/mL.

2) The chloroform/methanol was evaporated with a rotary evaporator,followed by vacuum-drying.

3) A 0.1N sodium hydroxide solution (>pH: 12.0) was added so that thelipid concentration was 10 mg/mL. The mixture was redispersed byvortexing, and subjected to ultrasonic treatment for 30 minutes in totalusing a VS-100III produced by AS ONE Corporation by repeating a cycle of28 kHz output for 60 seconds, 45 kHz output for 60 seconds, and 100 kHzoutput for 3 seconds. After the ultrasonic treatment, the particle sizewas confirmed.

4) To exchange the liposome external solution, stirred ultrafiltration(cutoff molecular weight: 300,000 Da) was performed. The liposomeexternal solution was a 10 mM phosphate buffer (pH: 8.0) (liposomesolution). For ultrafiltration, a stirred cell (8000 series, a 50-mLcell): Model 8050 5122 produced by Merck & Co., Inc. and BioMax PBMK04310 produced by Merck & Co., Inc. were used. After the preparation ofempty liposomes, lipid quantification was performed.

5) Compound (A) (56.5 mg) was weighed, and 1.41 mL of a 0.1N sodiumhydroxide solution was added thereto. The mixture was dissolved byvortexing, and 5.65 mL of a 10 mM phosphate buffer (pH: 8.0) was addedthereto to thus prepare a solution of 8 mg/mL (compound (A) solution).

6) The compound (A) solution was added to the liposome solution (threeconcentration conditions), and the mixtures were stirred at 60° C. for 1hour.

7) The three concentration conditions (the solution volume: 3 mL) were a1/10 mixing ratio: 3.29 mg of the compound and 32.9 mg of the lipids; a2/10 mixing ratio: 6.58 mg of the compound and 32.9 mg of the lipids;and a 4/10 mixing ratio: 13.16 mg of the compound and 32.9 mg of thelipids.

8) Free compound (A) was removed by ultrafiltration (cutoff molecularweight: 300,000 Da) using a stirred cell (8000 series, a 10-mL cell):Model 8010 5121 produced by Merck & Co., Inc., and BioMax PBMK 02510produced by Merck & Co., Inc. The external solution was a 10 mMphosphate buffer (pH: 7.4).

As a result, liposomal formulations having four different propertiesshown in Table 2 below were obtained. FIG. 1 is diagrams showing theaverage particle size distribution of these formulations.

TABLE 2 Average Zeta Lipid Concentration Compound particle sizepotential concentration of compound (A) (A)/lipid Content (Z-Ave, nm)PDI (mV) (mg/mL) (mg/mL) (mg/mL) (%) Formu- Blank 109 0.121 −67 8.44 — —— lation liposome 1 Formu- Mixing 105 0.105 −29 5.40 0.12 0.02 2.2lation ratio of 2 1/10 Formu- Mixing 102 0.126 −23 5.46 0.14 0.03 2.6lation ratio of 3 2/10 Formu- Mixing 103 0.148 −27 5.62 0.16 0.03 2.8lation ratio of 4 4/10 * Content = calculated from the concentration ofcompound (A) per mg of lipid. * Physical property testing was performedafter filtration through a 0.22 μm filter.

2. Formulation Example 2 (Bangham Method) Preparation of Liposome

1) HSPC (102.0 mg), DSPE (8.5 mg), and cholesterol (33.5 mg) wereweighed and placed in an eggplant flask, and a chloroform/methanolsolution (1/1, v/v) was added and dissolved so that the lipidconcentration was 20 mg/mL.

2) The chloroform/methanol was evaporated with a rotary evaporator,followed by vacuum-drying. Four eggplant flasks each containing 30 mg ofthe lipids in total were thus obtained.

3) Compound (A) (170.6 mg) was weighed, and 4.5 mL of a 0.1N sodiumhydroxide solution was added thereto. The mixture was dissolved byvortexing, and 2.6 ml of 10 mM phosphate buffer (pH: 8.0) was addedthereto to thus prepare a solution of 24 mg/mL (compound (A) solution).

4) The compound (A) solution was added to the vacuum-dried lipid film(three concentration conditions), followed by stirring at 37° C. for 1hour. Since each eggplant flask contained 30 mg of the lipids, theamount of each solution was adjusted with PBS to 3 mL to achieve a 1/10mixing ratio: 3.0 mg of compound (A) and 30 mg of the lipids; a 2/10mixing ratio: 6 mg of compound (A) and 30 mg of the lipids; or a 4/10mixing ratio: 12 mg of compound (A) and 30 mg of the lipids.

5) Treatment was performed for 60 minutes in total using a VS-100III,produced by AS ONE Corporation, by repeating a cycle of 28 kHz outputfor 60 seconds, 45 kHz output for 60 seconds, and 100 kHz output for 3seconds.

6) Ultrasonic treatment was performed for 60 minutes.

7) Free compound (A) was removed by ultrafiltration (cutoff molecularweight: 300,000 Da) using a stirred cell (8000 series, a 10-mL cell):Model 8010 5121 produced by Merck & Co., Inc., and BioMax PBMK 02510produced by Merck & Co., Inc.

The external solution was a 10 mM phosphate buffer (pH: 7.4).

As a result, liposomal formulations having four different propertiesshown in Table 3 below were obtained. FIG. 2 is diagrams showing theaverage particle size distribution of these formulations.

TABLE 3 Average Zeta Lipid Concentration Compound particle sizepotential concentration of compound (A) (A)/lipid Content (Z-Ave. nm)PDI (mV) (mg/mL) (mg/mL) (mg/mL) (%) Formu- Blank 75 0.172 −23 8.12 — —— lation liposome 5 Formu- Mixing 123 0.162 −30 6.47 0.07 0.011 1.1lation ratio of 6 1/10 Formu- Mixing 132 0.146 −32 6.76 0.08 0.012 1.2lation ratio of 7 2/10 Formu- Mixing 129 0.142 −32 6.66 0.21 0.032 3.2lation ratio of 8 4/10 * Content = calculated from the concentration ofcompound (A) per mg of lipid. * Physical property testing was performedafter filtration through a 0.22 μm filter.

3. Formulation Example 3 (Extruder Method) Preparation of Liposome

1) HSPC (255.0 mg), DSPE (21.3 mg), and cholesterol (83.0 mg) wereweighed and placed in an eggplant flask, and a chloroform/methanolsolution (1/1, v/v) was added and dissolved so that the lipidconcentration was 20 mg/mL.

2) The chloroform/methanol was evaporated with a rotary evaporator,followed by vacuum drying.

3) Compound (A) (143 mg) was weighed, and 3.76 mL of a 0.1N sodiumhydroxide solution was added thereto. The mixture was dissolved byvortexing, and 2.2 mL of a 10 mM phosphate buffer (pH: 8.0) was addedthereto to thus prepare a solution of 24 mg/mL (compound (A) solution).

4) The compound (A) solution was added to the lipid film, and a 10 mMphosphate buffer (pH: 8.0) was added to increase the volume to 36 mL(compound (A): 140 mg, the lipids: 360 mg).

5) Ultrasonic treatment was performed at 28 kHz for 1 minute using aVS-100III produced by AS ONE Corporation so that the lipids on the wallof the eggplant flask were dispersed.

6) At 60° C., heating and stirring were performed for 30 minutes.

7) An equivalent amount of 10 mM phosphate buffer (pH: 8.0) was added,and extruder treatment was performed (60° C., 400 nm, 200 nm). Theextruder was performed with a Lipex Thermobarrel Extruder (100 mL)produced by Northern Lipids, and with Nuclepore membranes produced by GEHealthcare (400 nm: Product No. 111107; 200 nm: Product No. 111106).

8) Free compound (A) was removed by ultrafiltration (cutoff molecularweight: 300,000 Da) using a stirred cell (8000 series, a 50-mL cell):Model 8050 5122 produced by Merck & Co., Inc., and BioMax PBMK 04310produced by Merck & Co., Inc.

The external solution was a 10 mM phosphate buffer (pH: 7.4).

As a result, a liposomal formulation having the properties shown inTable 4 below was obtained. FIG. 3 is a diagram showing the averageparticle size distribution of this formulation.

TABLE 4 Average Zeta Lipid Concentration Compound particle sizepotential concentration of compound (A) (A)/Lipid Content (Z-Ave. nm)PDI (mV) (mg/mL) (mg/mL) (mg/mL) (%) Formu- Mixing 175 0.147 −11 5.120.153 0.030 3.0 lation ratio of 9 4/10 * Content = calculated from theconcentration of compound (A) per mg of lipid. * Physical propertytesting was performed after filtration through a 0.22 μm filter.

4. Formulation Example 4 (Lipid-Compound Film Method) Preparation ofLiposome

1) Lipids (HSPC: 21.2 mg, DSPE: 1.8 mg, and cholesterol: 7.0 mg) andcompound (A) (6.0 mg) were weighed and placed in an eggplant flask. Atthis time, the amounts were adjusted so that compound A) was present inan amount of 2 mg per 10 mg of the lipids.

2) A chloroform/methanol solution (1/1, v/v) was added and dissolved sothat the total weight of the lipids and compound (A) was 20 mg/mL.

3) The chloroform/methanol was evaporated with a rotary evaporator,followed by vacuum-drying.

4) 10 mM phosphate buffer (pH 8.0) was added to the resulting product sothat the lipid concentration was 10 mg/mL.

5) Ultrasonic treatment was performed at 28 kHz using a VS-100IIIproduced by AS ONE Corporation to separate the lipids and compound (A)from the wall of the eggplant flask, and the mixture was stirred at 60°C. for 30 minutes.

6) An equivalent amount of 10 mM phosphate buffer (pH: 8.0) was addedthereto, and extruder treatment was performed (60° C., 400 nm, 200 nm).More specifically, the extruder was performed with a Lipex ThermobarrelExtruder (100 mL) produced by Northern Lipids, and with Nucleporemembranes produced by GE Healthcare (400 nm: Product No. 111107; and 200nm: Product No. 111106).

7) Free compound (A) was removed by ultrafiltration (cutoff molecularweight: 300,000 Da) using a stirred cell (8000 series, a 10-mL cell):Model 8010 5121 produced by Merck & Co., Inc., and BioMax PBMK 02510produced by Merck & Co., Inc. The external solution was a 10 mMphosphate buffer (pH: 7.4).

As a result, a liposomal formulation having the properties shown inTable 5 below was obtained. FIG. 4 is a diagram showing the averageparticle size distribution of this formulation.

TABLE 5 Average Zeta Lipid Concentration Compound particle sizepotential concentration of compound (A) (A)/lipid Content (Z-Ave. nm)PDI (mV) (mg/mL) (mg/mL) (mg/mL) (%) Formu- Mixing 158 0.132 −13 4.730.07 0.015 1.5 lation ratio of 10 2/10 * Content = calculated from theconcentration of compound (A) per mg of lipid. * Physical propertytesting was performed after filtration through a 0.22 μm filter.

5. Production of PEG-Treated Stealth Liposomal Formulation (1)Formulation Example 5 (Remote Loading Method) Preparation of Liposome

1) HSPC (107.5 mg), MPEG2000-DSPE (35.0 mg), and cholesterol (35.5 mg)were weighed and placed in an eggplant flask, and a chloroform/methanolsolution (1:1, v/v) was added and dissolved so that the lipidconcentration was 30 mg/mL.

2) The chloroform/methanol was evaporated with a rotary evaporator,followed by vacuum-drying. Then, a 0.1N sodium hydroxide solution (>pH:12.0) was added so that the lipid concentration was 10 mg/mL, and themixture was redispersed by vortexing.

3) Ultrasonic treatment was performed for 30 minutes in total using aVS-100III produced by AS ONE Corporation by repeating a cycle of 28 kHzoutput for 60 seconds, 45 kHz output for 60 seconds, and 100 kHz outputfor 3 seconds. Thereafter, the particle size was confined.

4) To exchange the liposome external solution, stirred ultrafiltration(cutoff molecular weight: 300,000 Da) was performed. The liposomeexternal solution was 10 mM phosphate buffer (pH: 8.0) (liposomesolution).

The ultrafiltration was performed using a stirred cell (8000 series, a50-mL cell): Model 8050 5122 produced by Merck & Co., Inc., and BioMaxPBMK 04310 produced by Merck & Co., Inc. After the preparation of emptyliposomes, lipid quantification was performed.

5) Compound (A) (56.5 mg) was weighed, and 1.41 mL of a 0.1N sodiumhydroxide solution was added thereto. The mixture was dissolved byvortexing, and 5.65 mL of 10 mM phosphate buffer (pH: 8.0) was addedthereto to thus prepare a solution of 8 mg/mL (compound (A) solution).

6) The compound (A) solution was added to the liposome solution (threeconcentration conditions), and the mixture was stirred at 60° C. for 1hour. The three concentration conditions (solution volume: 4 mL) were a1/10 mixing ratio: 4.0 mg of compound (A) and 40.0 mg of the lipids; a2/10 mixing ratio: 8.0 mg of compound (A) and 40.0 mg of the lipids; anda 4/10 mixing ratio: 16.0 mg of compound (A) and 40.0 mg of the lipids.

7) Free compound (A) was removed by stirred ultrafiltration (cutoffmolecular weight: 300,000 Da). Ultrafiltration was performed by using astirred cell (8000 series, a 10-mL cell): Model 8010 5121 produced byMerck & Co., Inc., and BioMax PBMK 02510 produced by Merck & Co., Inc.The external solution was a 10 mM phosphate buffer (pH: 7.4).

As a result, liposomal formulations having four different propertiesshown in Table 6 below were obtained. FIG. 5 is diagrams showing theaverage particle size distribution of these formulations.

TABLE 6 Average Zeta Lipid Concentration Compound particle sizepotential concentration of compound (A) (A)/lipid Content (Z-Ave. nm)PDI (mV) (mg/mL) (mg/mL) (mg/mL) (%) Formu- Blank 114 0.139 −23 5.94 — —— lation liposome 11 Formu- Mixing 119 0.113 −32 6.11 0.08 0.01 1.3lation ratio of 12 1/10 Formu- Mixing 120 0.096 −33 6.58 0.09 0.01 1.4lation ratio of 13 2/10 Formu- Mixing 116 0.131 −30 6.58 0.13 0.02 2.0lation ratio of 14 4/10 * Content = calculated from the concentration ofcompound (A) per mg of lipid. * Physical property testing was performedafter filtration through a 0.22 μm filter.

(2) Formulation Example 6 (Bangham Method) Preparation of Liposome

1) HSPC (87.0 mg), MPEG2000-DSPE (28.3 mg), and cholesterol (29.0 mg)were weighed and placed in an eggplant flask, and a chloroform/methanolsolution (1:1, v/v) was added and dissolved so that the lipidconcentration was 30 mg/mL.

2) The chloroform/methanol was evaporated with a rotary evaporator,followed by vacuum drying. Four eggplant flasks each containing 10 mg ofthe lipids in total were thus obtained.

3) Compound (A) (164 mg) was weighed, and 4.3 mL of a 0.1N sodiumhydroxide solution was added thereto. The mixture was dissolved byvortexing, and 2.53 mL of 10 mM phosphate buffer (pH: 8.0) was addedthereto to thus prepare a solution of 24 mg/mL (compound (A) solution).

4) The compound (A) solution was added to the vacuum-dried lipid film(three concentration conditions), followed by stirring at 37° C. for 1hour.

5) The three concentration conditions (solution volume: 4 mL) were a1/10 mixing ratio: 1.0 mg of compound (A) and 10.0 mg of the lipids; a2/10 mixing ratio: 2.0 mg of compound (A) and 10.0 mg of the lipids; anda 4/10 mixing ratio: 4.0 mg of compound (A) and 10.0 mg of the lipids.

6) Ultrasonic treatment was performed for 60 minutes in total using aVS-100III produced by AS ONE Corporation by repeating a cycle of 28 kHzoutput for 60 seconds, 45 kHz output for 60 seconds, and 100 kHz outputfor 3 seconds.

7) Free compound (A) was removed by ultrafiltration (cutoff molecularweight: 300,000 Da) using a stirred cell (8000 series, a 10-mL cell):Model 8010 5121 produced by Merck & Co., Inc., and BioMax PBMK 02510produced by Merck & Co., Inc. The external solution was a 10 mMphosphate buffer (pH: 7.4).

As a result, liposomal formulations having four different propertiesshown in Table 7 below were obtained. FIG. 6 is diagrams showing theaverage particle size distribution of these formulations.

TABLE 7 Average Lipid particle Zeta concen- Concentration Compound sizepotential tration of compound (A)/lipid Content (Z-Ave. nm) PDI (mV)(mg/mL) (A) (mg/mL) (mg/mL) (%) Formu- Blank 115 0.205 −48 7.58 — — —lation liposome 15 Formu- Mixing 95 0.213 −45 8.57 0.07 0.009 0.9 lationratio of 16 1/10 Formu- Mixing 110 0.243 −48 6.93 0.17 0.024 2.4 lationratio of 17 2/10 Formu- Mixing 102 0.221 −43 7.77 0.27 0.035 3.5 lationratio of 18 4/10 * Content = calculated from the concentration ofcompound (A) per mg of lipid. * Physical property testing was performedafter filtration through a 0.22 μm filter.

(3) Formulation Example 7 (Extruder Method) Preparation of Liposome

1) HSPC (215.0 mg), MPEG2000-DSPE (70.0 mg), and cholesterol (71.0 mg)were weighed and placed in an eggplant flask, and a chloroform/methanolsolution (1:1, v/v) was added and dissolved so that the lipidconcentration was 30 mg/mL.

2) The chloroform/methanol solution was evaporated with a rotaryevaporator, followed by vacuum-drying.

3) Compound (A) (142.7 mg) was weighed, and 3.8 mL of a 0.1N sodiumhydroxide solution was added thereto. The mixture was dissolved byvortexing, and 2.19 mL of 10 mM phosphate buffer (pH: 8.0) was addedthereto to thus prepare a solution of 24 mg/mL (compound (A) solution).

4) The compound (A) solution was added to the lipid film, and 10 mMphosphate buffer (pH: 8.0) was added thereto to increase the volume to35.6 mL (compound (A): 142.4 mg, the lipids: 356.0 mg).

5) Ultrasonic treatment was performed for 1 minute and for 30 minutes intotal using a VS-1001 produced by AS ONE Corporation by repeating acycle of 28 kHz output for 60 seconds, 45 kHz output for 60 seconds, and100 kHz output for 3 seconds to separate the lipids from the wall of theeggplant flask, and the resulting product was stirred at 60° C. for 30minutes.

6) An equivalent amount of 10 mM phosphate buffer (pH: 8.0) was addedthereto, and extruder treatment was performed (60° C., 400 nm, 200 nm).The extruder was performed with a Lipex Thermobarrel Extruder (100 mL)produced by Northern Lipids, and with Nuclepore membranes produced by GEHealthcare (400 nm: Product No. 111107; 200 nm: Product No. 111106).

7) Free compound (A) was removed by ultrafiltration (cutoff molecularweight: 300,000 Da) using a stirred cell (8000 series, a 50-mL cell):Model 8050 5122 produced by Merck & Co., Inc., and BioMax PBMK 04310produced by Merck & Co., Inc. The external solution was a 10 mMphosphate buffer (pH: 7.4).

As a result, a liposomal formulation having the properties shown inTable 8 below was obtained. FIG. 7 shows is diagrams showing the averageparticle size distribution of this formulation.

TABLE 8 Average Lipid particle Zeta concen- Concentration Compound sizepotential tration of compound (A)/lipid Content (Z-Ave. nm) PDI (mV)(mg/mL) (A) (mg/mL) (mg/mL) (%) Formu- Mixing 158 0.114 −35 5.03 0.1280.025 2.5 lation ratio of 19 4/10 * Content = calculated from theconcentration of compound (A) per mg of lipid. * Physical propertytesting was performed after filtration through a 0.22 μm filter.

(4) Formulation Example 8 (Lipid-Compound Film Method) Preparation ofLiposome

1) HSPC (18.0 mg), MPEG2000-DSPE (6.0 mg), cholesterol (6.0 mg), andcompound (A) (6.0 mg) were weighed and placed in an eggplant flask. Atthis time, the amounts were adjusted so that compound (A) was present inan amount of 2 mg per 10 mg of the lipids. Then, a chloroform/methanolsolution (1/1, v/v) was added and dissolved so that the total weight ofthe lipids and compound (A) was 30 mg/mL.

2) The chloroform/methanol was evaporated with a rotary evaporator,followed by vacuum-drying.

3) 10 mM phosphate buffer (pH 8.0) was added to the resulting product sothat the lipid concentration was 10 mg/mL.

4) Ultrasonic treatment was performed at 28 kHz using a VS-100IIIproduced by AS ONE Corporation to separate the lipids and compound (A)from the wall of the eggplant flask, and the mixture was stirred at 60°C. for 30 minutes.

5) An equivalent amount of 10 mM phosphate buffer (pH: 8.0) was addedthereto, and extruder treatment was performed (60° C., 400 nm, 200 nm).The extruder was performed with a Lipex Thermobarrel Extruder (100 mL)produced by Northern Lipids and with Nuclepore membranes produced by GEHealthcare (400 nm: Product No. 111107; 200 nm: Product No. 111106).

6) Free compound (A) was removed by ultrafiltration (cutoff molecularweight: 300,000 Da) using a stirred cell (8000 series, a 10-mL cell):Model 8010 5121 produced by Merck & Co., Inc., and BioMax PBMK 02510produced by Merck & Co., Inc. The external solution was a 10 mMphosphate buffer (pH: 7.4).

As a result, Liposomal Formulation 20 having the properties shown inTable 9 below was obtained. FIG. 8 is a diagram showing the averageparticle size distribution of this formulation.

TABLE 9 Average Lipid particle Zeta concen- Concentration Compound sizepotential tration of compound (A)/lipid Content (Z-Ave. nm) PDI (mV)(mg/mL) (A) (mg/mL) (mg/mL) (%) Formu- Mixing 135 0.136 −30 11.30 0.060.005 0.5 lation ratio of 20 2/10 * Content = calculated from theconcentration of compound (A) per mg of lipid. * Physical propertytesting was performed after filtration through a 0.22 μm filter.

6. Method for Quantifying Compound (A)

1) Compound (A) (5 mg) was weighed and placed in a test tube, and a 0.1Nsodium hydroxide solution (0.125 mL) was added. The resulting mixturewas dissolved by vortexing.

2) Purified water (4.875 mL) was added thereto to thus prepare acompound (A) solution of 1 mg/mL.

3) A calibration curve was prepared by measuring the absorbance atwavelength of 265 nm with respect to 6 different compound (A)concentrations of 0.0 mg/mL to 0.50 mg/mL.

4) Compound (A) solutions of known concentrations (0.1 mg/mL, 0.2 mg/mL,and 0.4 mg/mL) were prepared, and sample concentrations were obtainedfrom the calibration curve.

5) The compound (A) solutions of known concentrations (0.1 mg/mL, 0.2mg/mL, and 0.4 mg/mL) were each added to blank liposome (lipidconcentration: 4.7 mg/mL), and the compound (A) concentrations weremeasured.

6) The recovery rates were calculated from the measurement results ofthe known concentration solutions, and the measurement results of theknown concentration solutions to which liposome was added.

Table 10 and FIG. 9 show the measurement results.

TABLE 10 Standard Curve Chloroform/methanol solution Compound AAbsorbance 265 nm (mg/mL) {circle around (1)} {circle around (2)}{circle around (3)} Average 0.000 0.129 0.130 0.127 0.000 0.031 0.2930.291 0.291 0.163 0.063 0.435 0.450 0.450 0.316 0.125 0.736 0.761 0.7610.624 0.250 1.360 1.374 1.381 1.243 0.500 2.567 2.587 2.642 2.470 Slope4.9321 Intercept 0.0064 Recovery Concentration rate Recovery rate{circle around (1)} {circle around (2)} {circle around (3)} Average(mg/ml) (%) (−Liposome) Single drug: 0.588 0.597 0.613 0.503 0.101 1010.1 mg/ml Single drug: 0.966 0.925 0.998 0.867 0.174 87 0.2 mg/ml Singledrug: 1.887 1.911 1.922 1.810 0.366 91 0.4 mg/ml A) Liposome: 0.1460.146 0.148 0.050 0.009 0.0 mg A) Liposome: 0.591 0.600 0.604 0.5020.200 100 91 0.1 mg A) Liposome: 1.058 1.056 1.054 0.960 0.193 111 1060.2 mg A) Liposome: 1.881 1.922 1.936 1.817 0.367 100 98 0.4 mg B)Liposome: 0.141 0.141 0.144 0.046 0.008 0.0 mg B) Liposome: 0.580 0.5910.581 0.488 0.098 97 89 0.1 mg B) Liposome: 1.000 1.027 1.011 0.9160.284 106 101 0.2 mg B) Liposome: 1.845 1.866 1.889 1.770 0.358 98 980.4 mg

The results confirmed that no effect was caused by the lipids formedinto liposomes; thus, compound (A) was quantified by using an absorbancemethod.

7. Mass Production of Nanosphere of Stealth Liposome of Compound (A) byBangham Method

(1) A PEG-treated compound (A)-encapsulated liposome was prepared inlarge quantities by the Bangham method, and the properties were tested.

Production Method

(i) HSPC (5.524 g), cholesterol (1.8419 g), and MPEG2000-DSPE (1.7978 g)were weighed and placed in an eggplant flask, a chloroform/methanol(1/1, v/v) solution was added so that the lipid concentration was 30mg/mL, and the mixture was dissolved by stirring at 37° C.

(ii) The solvent was distilled off with an evaporator in a nitrogenatmosphere, followed by vacuum-drying. Fifteen 100-ml eggplant flaskseach containing 600 mg of the lipids and one 100-ml eggplant flaskcontaining 300 mg of the lipids were thus prepared.

(iii) Compound (A) was weighed, and a 0.1N sodium hydroxide solution wasadded thereto to produce a solution having a compound (A) concentrationof 40 mg/mL. 10 mM phosphate buffer (pH: 8.0) was added to the solutionso that the compound (A) concentration was 24 mg/mL.

(iv) The solution obtained in (iii) was added to the lipid film; and 10mM phosphate buffer (pH: 8.0) was added thereto so that the lipidconcentration was 10 mg/mL, followed by stirring at 37° C. for 1 hour.Then, 22.2 mg of compound (A) was added to the eggplant flasks eachcontaining 600 mg of the lipids, and 11.1 mg of compound (A) was addedto the flask containing 300 mg of the lipids.

(V) Ultrasonic treatment was performed for each of the eggplant flasksfor 60 minutes in total using a VS-100III produced by AS ONE Corporationby repeating a cycle of 28 kHz output for 60 seconds, 45 kHz output for60 seconds, and 100 kHz output for 3 seconds.

(vi) A stirred cell (8000 series, a 400-mL cell): Model 8400 5124produced by Merck & Co., Inc., and BioMax PBMK 07610 produced by Merck &Co., Inc. were used. Since the solution amount was 930 mL in total,three cells were used for ultrafiltration (cutoff molecular weight:300,000 Da) to replace the outer aqueous phase with 10 mM phosphatebuffer (pH 7.4), and unencapsulated compound (A) was removed.

As a result, a liposomal formulation having the properties shown inTable 11 below was obtained. FIG. 10 is a diagram showing the averageparticle size distribution of this formulation.

TABLE 11 Average Lipid particle Zeta concen- Concentration Compound sizepotential tration of compound (A)/lipid Content (Z-Ave. nm) PDI (mV)(mg/mL) (A) (mg/mL) (mg/mL) (%) Formu- Large-scale 163 0.265 −58 72.22.03 0.028 2.8 lation preparation 21 * Content = calculated from theconcentration of compound (A) per mg of lipid. * Physical propertytesting was performed after filtration through a 0.22 μm filter.

The following general test was conducted using part of the producedPEG-treated compound (A)-encapsulated liposome.

(2) FIG. 11 is transmission electron microscope images of Formulation21.

Analysis method: Morphological observation

Photographing device: Hitachi H-7600 at 100 kV

Sample production method

-   -   Dispersion: 400-mesh grid with carbon support membrane    -   Staining: Negative staining (phosphotungstic acid)

FIG. 11 shows electron micrographs.

(3) Part of the produced PEG-treated compound (A)-encapsulated liposomewas subjected to HPLC analysis to confirm the presence or absence ofdecomposition products.

-   -   Analysis and quantification of compound (A) by HPLC

Apparatus: Agilent 1290 Infinity LC series

Column: Shiseido Capcell Pak C18 UG120, 5 μm, 4.6×150 mm

Mobile phase: 1% acetic acid/H₂O: 1% acetic acid/acetonitrile=60:40

Flow rate: 1 mL/min

Detection wavelength: 265 nm

Column temperature: 25° C.

Sample Preparation Method

Sample 1: Compound (A) was dissolved in a 1M sodium hydroxide solutionso that compound A was 1 mg/mL. The resulting product was allowed tostand at 40° C. overnight (oxide production sample).

Sample 2: Compound (A) was added to a 0.1N sodium hydroxide solution-10mM phosphate buffer (pH: 8.0) (buffer for encapsulation) so thatcompound (A) was 0.5 mg/mL (control sample)

Sample 3: Sample 2 was subjected to two-fold dilution with physiologicalsaline to prepare a solution of 0.25 mg/mL. The solution was allowed tostand at 37° C. overnight (compound control sample).

Sample 4: PEG-treated empty liposomes were diluted 10-fold withphysiological saline. The resulting product was allowed to stand at 37°C. overnight, followed by ultrafiltration (lipid control sample).

Sample 5: The PEG-treated compound (A)-encapsulated liposome was diluted10-fold with physiological saline. The resulting product was allowed tostand at 37° C. overnight, followed by ultrafiltration (liposomesample).

The above 5 samples were analyzed by HPLC to confirm the presence orabsence of oxides of compound (A), and the presence or absence of oxidesof compound (A) of the PEG-treated compound (A)-encapsulated liposome inan environment at a temperature of 37° C.

FIG. 12 charts of HPLC measurement of Samples 1 to 5.

8. Production of PEG-Treated Compound (B)-Encapsulated Stealth Liposomeby Bangham Method

(1) Production of Liposome

(i) HSPC (36.0 mg), cholesterol (12.0 mg), and MPEG2000-DSPE (12.0 mg)were weighed and placed in an eggplant flask, a chloroform/methanol(1/1, v/v) solution was added so that the lipid concentration was 30mg/mL, and the mixture was dissolved by stirring at 37° C.

(ii) The solvent was distilled off with an evaporator in a nitrogenatmosphere, followed by vacuum-drying.

(iii) Compound (B) was added to and dissolved in 10 mM phosphate buffer(pH: 8.0) so that compound (B) was 20 mg/mL.

(iv) The compound (B) solution was added to the lipid film to achieve1/10 (drug/lipid, w/w %), and 10 mM phosphate buffer (pH: 8.0) was addedso that the lipid concentration was 10 mg/mL (compound (B): 6.0 mg, thelipids: 60.0 mg).

(v) The resulting product was vortexed for about 20 seconds, followed bystirring at 37° C. for 1 hour.

(vi) Ultrasonic treatment was performed for 90 minutes in total using aVS-100III produced by AS ONE Corporation by repeating a cycle of 28 kHzoutput for 60 seconds, 45 kHz output for 60 seconds, and 100 kHz outputfor 3 seconds.

(vii) A stirred cell (8000 series, a 10-mL cell): Model 8010 5121produced by Merck & Co., Inc., and BioMax PBMK 02510 produced by Merck &Co., Inc. were used. The amount was 6 mL at the time of start and 4 mLat the time of collection, and 1.5-fold concentration was performed.Since the drug encapsulation amount was low, ultrafiltration (cutoffmolecular weight: 300,000 Da) was performed to replace the outer aqueousphase with 10 mM phosphate buffer (pH 7.4), and unencapsulated compound(B) was removed.

As a result, a liposomal formulation having the properties shown inTable 12 below was obtained. FIG. 13 is a diagram showing the averageparticle size distribution of this formulation.

TABLE 12 Average Lipid particle Zeta concen- Concentration Compound sizepotential tration of compound (B)/lipid Content (Z-Ave. nm) PDI (mV)(mg/mL) (B) (mg/mL) (mg/mL) (%) Formu- Mixing 87 0.199 −32 12.19 0.1010.008 0.8 lation ratio of 22 1/10 * Content = calculated from theconcentration of compound (A) per mg of lipid. * Physical propertytesting was performed after filtration through a 0.22 μm filter.

Accordingly, a PEG-treated compound (B)-encapsulated liposome wasproduced.

(2) FIG. 14 is transmission electron microscope images of Formulation22.

(3) Analysis of Method of Quantifying Compound (B)

Absorption Spectrum Confirmation Method

(i) Compound (B) was added to and dissolved in 10 mM phosphate buffer(pH: 8.0) so that compound (B) was 20 mg/mL.

(ii) The product obtained in (i) was subjected to two-fold, four-serialdilutions with purified water.

(iii) The sample of each concentration was diluted 10-fold with achloroform/methanol (1/1, v/v) solution, and the absorption spectrum wasmeasured at 220 to 700 nm using UV2700.

The absorption spectrum of compound (B) was measured. As a result, themaximum absorption wavelength was 285 nm. It was confirmed that thequantification of compound (B) could be measured by the absorbancemethod (absorbance: 285 nm). FIG. 15 is a graph showing changes in theUV absorption spectrum.

9. Production of PEG-Treated Compound (C)-Encapsulated Stealth Liposomeby Bangham Method

(1) Liposome Production Method

(i) HSPC (60.0 mg), cholesterol (12.0 mg), and MPEG2000-DSPE (12.0 mg)were weighed and placed in an eggplant flask, a chloroform/methanol(1/1, v/v) solution was added so that the lipid concentration was 30mg/mL, and the mixture was dissolved by stirring at 37° C.

(ii) The solvent was distilled off with an evaporator in a nitrogenatmosphere, followed by vacuum-drying.

(iii) Compound (C) was dissolved in DMSO so that compound (C) was 20mg/mL, and 10 mM phosphate buffer (pH: 8.0) was added so that compound(C) was 10 mg/mL.

(iv) The compound (C) solution was added to the lipid film to achieve1/10 (drug/lipid, w/w %), and the mixture was stirred at 37° C. for 1hour (compound (C): 6.0 mg, the lipids: 60.0 mg).

(v) Ultrasonic treatment was performed for 90 minutes in total using aVS-100III, produced by AS ONE Corporation by repeating a cycle of 28 kHzoutput for 60 seconds, 45 kHz output for 60 seconds, and 100 kHz outputfor 3 seconds.

(vi) Ultrafiltration (cutoff molecular weight: 300,000 Da) was performedusing a stirred cell (8000 series, a 10-mL cell): Model 8010 5121produced by Merck & Co., Inc., and BioMax PBMK 02510 produced by Merck &Co., Inc. to replace the outer aqueous phase with 10 mM phosphate buffer(pH 7.4), and unencapsulated compound (C) was removed.

(2) As a result, a liposome having the properties shown in Table 13 wasobtained. FIG. 16 is a diagram showing the average particle sizedistribution.

TABLE 13 Average Lipid particle Zeta concen- Concentration Compound sizepotential tration of compound (C)/lipid Content (Z-Ave. nm) PDI (mV)(mg/mL) (C) (mg/mL) (mg/mL) (%) Formu- Mixing 97 0.158 −34 17.27 1.020.059 5.9 lation ratio of 23 1/10 * Content = calculated from theconcentration of compound (A) per mg of lipid * Physical propertytesting was performed after filtration through a 0.22 μm filter.

Accordingly, a PEG-treated compound (C)-encapsulated liposome wasprepared.

(3) FIG. 17 is transmission electron microscope images of Formulation23.

(4) Analysis of Method of Quantifying Compound (C)

Absorption Spectrum Confirmation Method

(i) Compound (C) was added to and dissolved in DMSO so that compound (C)was 20 mg/mL.

(ii) An equivalent amount of 10 mM phosphate buffer (pH: 8.0) was addedto the product obtained in (i) to produce a 50% DMSO solution having acompound (C) concentration of 10 mg/mL.

(iii) The product obtained in (ii) was subjected to two-fold,three-serial dilution with purified water.

(vi) The sample of each concentration was diluted 10-fold with achloroform/methanol (1/1, v/v) solution, and the absorption spectrum wasmeasured at 220 to 700 nm using UV2700.

The absorption spectrum of compound (C) was measured; the absorptionpeaks were detected at 300 nm and 368 nm. It was confirmed that thequantification of compound (C) could be performed by the absorbancemethod (wavelength: 300 nm). FIG. 18 is a graph showing changes in theUV absorption spectrum of compound (C).

10. Production of Stealth Liposomal Formulation of Compound (A)(ONO-1301) by Direct Dispersion Improving Method

1) Production Method

(i) The two types of lipids and compound (A) were weighed as shown inTable 14 below, and dissolved in 20 g of t-butanol at 70° C.

TABLE 14 Composition 1 Composition 2 Lipid or API (Formulation 24)(Formulation 25) DEPC 1.88 g 1.80 g MPEG2000-DSPE 0.12 g 0.20 g Compound(A) 100 mg 100 mg

(ii) The liquid obtained by dissolution in (i) was instantly frozen indry ice/acetone.

(iii) After freezing, the resulting product was freeze-dried for about17 hours with a freeze-dryer.

(iv) 440 mL of PBS(−) was added to the obtained powder, and the mixturewas placed in a warm bath at 50° C. and dispersed by a sonicator untilno lumps were present (at this time, the ONO-1301 concentration was 2.5mg/mL).

(v) A polycarbonate filter with a pore size of 400 nm and a drain discwere attached, and sizing was performed at about 50 kg/cm² with anextruder in which the jacket was watered with warm water at 50° C.

(vi) In the same manner as (v), sizing was performed with a 200-nmfilter (3 Pass) to thus obtain a translucent liposome solution.

2) Ultrafiltration

Ultrafiltration (ultrafiltration membrane: PBMK04310, Merck Millipore,cutoff molecular weight: 300,000 Da) was performed using PBS(−) (10-folddilution of SIGMA D1408) to remove unencapsulated compounds.

Ultrafiltration was performed using a stirred cell, 8000 series: Model8050, product No. 5122, produced by Merck & Co., Inc. For the membrane,BioMax PBMK 04310,300 kDa, produced by Merck & Co., Inc., was used.

After the filtration, sterilization and filtration with a 0.22-μm filterwas performed in a clean bench.

3) Confirmation of Properties

The liposome solution was subjected to ultrafiltration (cutoff molecularweight: 300,000 Da) with PBS(−): Dulbecco's phosphate buffered saline(without Ca and Mg), and the resulting product was used as a sampleafter ultrafiltration. Additionally, the presence or absence of oxidesof ONO-1301 was confirmed by HPLC.

As shown in Table 15, the results showed no significant change in eachphysical property before and after the ultrafiltration. Additionally, asshown in FIGS. 19 and 20, no peaks of ONO-1301 decomposition productswere observed in the particle size distribution or the HPLC analysisresults. The yield of compound (A) was 88%.

4) Stability Test

After ultrafiltration (Formulation 25), the stability was analyzed afterstorage at 4° C. for 9 months using PBS(−): Dulbecco's phosphatebuffered saline (without Ca and Mg); the results confined no change inthe particle size distribution, content, and other physical properties;thus, the formulation was confirmed to be stable.

TABLE 15 Compound Average Lipid Compound of the particle Zeta concen- ofthe invention/ size potential tration invention lipid Content (Z-Ave.nm) PDI (mV) (mg/mL) (mg/mL) (mg/mL) (%) Formu- Before 91.4 0.16 −3.441.8 2.5 0.060 6.0 lation ultrafil- 24 tration Formu- After 86.8 0.14−2.1 40.1 2.2 0.055 5.5 lation ultrafil- 25 tration

5) Confirmation of Decomposition Product (HPLC Analysis)

The HPLC measurement results confirmed that no decomposition products ofcompound (A) were present under the conditions of this production.

11. Production of Stealth Liposomal Formulations of Compound (B),Compound (D), and Compound (E) by Direct Dispersion Improving Method

Liposome Production Method

(i) DEPC (Nippon Fine Chemical Co., Ltd.) (0.188 g) and MPEG2000-DSPE(Nippon Fine Chemical Co., Ltd.) (0.012 g), and compound (B, D, or E)(0.01 g) were weighed and placed in a glass vial.

(ii) 2.0 g of t-butanol was added to the product obtained in (i), andthe mixture was dissolved by stirring while heating at 37° C.

(iii) The resulting product was instantly frozen in ethanol-dry ice.

(iv) Freeze-drying was performed for 17 hours.

(v) PBS(−) (Dulbecco's phosphate buffered saline (without Ca and Mg))(20 mL) was added to the lipid/compound powder obtained afterfreeze-drying.

(vi) After the resulting product was vortexed lightly, ultrasonictreatment was performed for 30 minutes while heating at 37° C.

(vii) Extruder treatment was performed while heating at 37° C. For theapparatus, a LIPEX extruder (100 mL) was used. After treatment wasperformed once with a polycarbonate filter with a pore size of 400 nm,treatment was performed three times with a polycarbonate filter with apore size of 200 nm (treatment pressure: about 5 MPa). The extruder usedwas a Lipex Thermobarrel Extruder (100 mL) produced by Northern Lipids,and the membranes used were Nuclepore membranes produced by GEHealthcare (400 nm: Product No. 111107; 200 nm: Product No. 111106).

(viii) Ultrafiltration (ultrafiltration membrane: PBMK04310, MerckMillipore, cutoff molecular weight: 300,000 Da) was performed usingPBS(−) (10-fold dilution of SIGMA D1408), and unencapsulated compoundswere removed. The ultrafiltration was performed until theultrafiltration waste liquid became 140 mL, which was 7 times theliposome amount (20 mL); and the resulting product in the final amountof 20 mL was collected.

The ultrafiltration was performed using a stirred cell, 8000 series:Model 8050, product No. 5122, produced by Merck & Co., Inc. For themembrane, BioMax PBMK 04310, 300 kDa, produced by Merck & Co., Inc., wasused. The liposome solution was introduced into the stirred cell, theapparatus was set, and feeding of the solution was performed by nitrogengas pressurization (0.4 MPa) until 140 mL of waste liquid wasdischarged.

(ix) Sterilization and filtration were performed with a 0.22-μm filterin a clean bench.

(x) A physical property test was performed. In the physical propertytest, the average particle size measurement, zeta potential measurement,lipid quantification (Wako Pure Chemical Industries, Phospholipid C-TestWako), and absorbance measurement (Abs 680 nm) were performed.

Test Results (1) Production of Compound (B) (Formulation26)-Encapsulated Liposome

The compound (B)-encapsulated liposome had the physical properties shownin Table 16 and FIG. 21. As shown in FIG. 22, UV absorption derived fromcompound (B) was observed in the UV absorption spectrum of the compound(B)-encapsulated liposome. Table 16 below shows the properties (theparticle size, PdI value, and zeta potential) of the obtained liposome.FIG. 21 shows the liposome particle size distribution of Formulation 26.

FIG. 22 shows UV absorption spectra of compound (B) (Beriplast) andliposomes containing the compound (B).

TABLE 16 Lipid Encapsulated Average particle Zeta potential (mg/mL)amount (mg/mL) size (nm) PdI (mV) 6.2 0.017 or less 122 0.190 −19

(2) Production of Compound (D) (Formulation 27)-Encapsulated StealthLiposome

The amount of encapsulated compound (D) was 0.249 mg/mL according to UVquantification. The obtained liposome had the properties shown in Table17 below; i.e., the particle size, PdI value, and zeta potential. FIG.23 shows the liposome particle size distribution of Formulation 27.

The absorption spectrum of FIG. 24 shows quantitativity at UV absorptionof 233 nm. FIG. 24 is UV absorption spectra of compound (D) (Limaprost)and liposomes containing Limaprost.

TABLE 17 Formulation 27: Lipid Encapsulated Average particle Zetapotential (mg/mL) amount (mg/mL) size (nm) PdI (mV) 6.0 0.249 110 0.200−19

(3) Production of Compound (E) (Formulation 28)-Encapsulated StealthLiposome

A compound (E)-encapsulated liposome had the physical properties shownin Table 18 and FIG. 25. The amount of encapsulated compound (E) was0.305 mg/mL according to UV quantification. The table below shows theparticle size, PdI value, and zeta potential. The absorption spectra ofFIG. 26 shows quantitativity at UV absorption of 300 nm.

TABLE 18 Formulation 28: Lipid Encapsulated Average particle Zetapotential (mg/mL) amount (mg/mL) size (nm) PdI (mV) 5.9 0.305 131 0.171−36

11. Various Pharmacodynamic and Pharmacological Tests Using Compound (A)Stealth Liposomes of Formulations 21 and 25 1) Analysis of Effect ofIntermittent Intravenous Administration of Formulation 21 (ONO-1301Lipo)on Rat Monocrotaline-Induced Pulmonary Hypertension Model

The produced Formulation 21 (ONO-1301Lipo formulation) as a testsubstance was intermittently administered intravenously once weekly to arat monocrotaline (MCT)-induced severe heart failure (pulmonaryhypertension) model from day 7 of the MCT administration, and thesurvival rate was compared with a group in which compound (A) (ONO-1301)was repeatedly orally administered; a group in which compound (A) wasintermittently administered intravenously once weekly; and, as apositive control, a group in which an ET-1 antagonist (bosentan) wasorally administered. In the control group, physiological saline(vehicle) was administered once weekly by intravenous administration.

For animals, Slc: Wistar male rats, 5 weeks old at the start of thetest, and 66.4 to 90.8 g at arrival (Japan SLC, Inc.) were used.Monocrotaline (hereinafter referred to as “MCT”), lot No.: SLBG1999V(Sigma-Aldrich Corporation), was administered once subcutaneously at theback of the rats at a dose of 60 mg/kg. Six days after the MCTadministration, the animals were divided into groups according to theweight stratification assignment method (Table 19).

TABLE 19 Administered substance/ dose/frequency of Route of Number ofGroup administration administration cases 1 Physiological saline/weekIntravenous 20 injection 2 Compound (A) (ONO-1301), Oral 10 3 mg/kg ×twice/day administration 3 Bosentan, 50 mg/kg × twice/day Oral 10administration 4 Formulation 21.1 mg/kg/week* Intravenous 10 injection 5Compound (A) (ONO-1301), Intravenous 10 1 mg/kg/week injection *The doseis in terms of compound (A) (ONO-1301).

Group 1: Physiological saline was intravenously administered 7, 14, 21,28, and 35 days after the MCT administration at weekly intervals (5times in total).

Group 2: Compound (A) was orally administered at 3 mg/kg twice dailyfrom 7 days to 41 days after the MCT administration with administrationintervals of 8 hours or more.

Group 3: Bosentan was orally administered at 50 mg/kg twice daily from 7days to 41 days after the MCT administration with administrationintervals of 8 hours or more.

Group 4: Formulation 21 (ONO-1301Lipo) was intravenously administered at1 mg/kg 7, 14, 21, 28, and 35 days after the MCT administration atweekly intervals (5 times in total).

Group 5: Compound (A) was intravenously administered at 1 mg/kg 7, 14,21, 28, and 35 days after the MCT administration at weekly intervals (5times in total).

FIG. 27 shows changes in the survival rates of Group 1, Group 2, andGroup 4 until 42 days after the preparation of the severe heart failuremodel. Table 20 shows the survival rates of all of the groups after 42days.

In Group 1, one death was observed 15 days after the preparation of thesevere heart failure model by subcutaneous administration of MCT at 60mg/kg. Thereafter, 17 deaths were observed by day 42, and the finalsurvival rate was 10% (number of animals alive: 2/20).

In the group in which compound (A) was repeatedly orally administered at3 mg/kg twice daily (Group 2), one death was observed 14 days after thepreparation of the severe heart failure model. Thereafter, 4 more deathswere observed by day 42, and the final survival rate was 50% (number ofanimals alive: 5/10), showing a significant life-prolonging effectcompared with the control group (Group 1) (p<0.05).

In the group in which bosentan was repeatedly orally administered at 50mg/kg twice daily (Group 3), one death was observed 20 days after thepreparation of the severe heart failure model. Thereafter, another 6deaths were observed by day 42, and the final survival rate was 30%(number of animals alive: 3/10), showing no significant life-prolongingeffect compared with the control group (Group 1).

Bosentan, which is an ET-1 antagonist, used in the positive control, hasbeen clinically used as a therapeutic agent for pulmonary hypertension,and the efficacy thereof has been confirmed in the same rat MCT-inducedheart failure model (Circ. J. 2013; 77: 2127-2133). However, theseresults are based on repeated oral administration immediately after theMCT administration. This time, a significant life-prolonging effectcould not be observed in the administration started 7 days after the MCTadministration (Group 3).

In the group in which Formulation 21 (ONO-1301Lipo) was intermittentlyadministered intravenously at 1 mg/kg once weekly (Group 4), one deathwas observed 27 days after the preparation of the severe heart failuremodel. Thereafter, 4 deaths were observed by day 42, and the finalsurvival rate was 50% (number of animals alive: 5/10), showing asignificant life-prolonging effect compared with the control group(Group 1) (p<0.05).

In the group in which compound (A) was intermittently administeredintravenously at 1 mg/kg once weekly (Group 6), two deaths were observedabout 30 minutes after the administration 28 days after the preparationof the severe heart failure model. Thereafter, 6 deaths were observed byday 42, and the final survival rate was 20% (number of animals alive:2/10), showing no significant life-prolonging effect compared with thecontrol group (Group 1).

TABLE 20 Administered substance, Number Survival dose, frequency ofRoute of of rate Group administration administration cases (%) 1Physiological saline Intravenous 20 10 injection 2 Compound (A) (ONO-Oral 10  50* 1301); 3 mg/kg × twice/day administration 3 Bosentan; 50mg/kg × Oral 10 30 twice/day administration 4 Formulation 21;Intravenous 10  50* 1 mg/kg/week injection 5 Compound (A) (ONO-Intravenous 10 20 1301); 1 mg/kg/week injection

The total test substance amount of compound (A) administered from 7 daysto 41 days after the MCT administration (35 days in total) wascalculated per animal.

As a result, the amount was 210 mg/kg/animal (3 mg/kg×twice/day×35 days)in Group 2, 3500 mg/kg/animal (50 mg/kg×twice/day×35 days) in Group 3,and 5 mg/kg/animal (1 mg/kg/week×5 times) in Group 4 and Group 5. Group4 showed an effect similar to that of Group 2 at a 5/210 (1/42) doseamount of Group 2.

As a disease site-specific DDS liposomal formulation, a novel ONO-1301liposomal formulation (Formulation 21; ONO-1301Lipo) was produced toanalyze the development of a therapeutic method for developing a moreversatile, disease site-specific (DDS) therapeutic agent for a severeheart failure, by intermittent intravenous administration.

The DDS effect was confirmed by comparing the survival rates in theMCT-induced severe heart failure model by the intermittent intravenousadministration of Formulation 21.

Bosentan, which is an ET-1 antagonist, and used as the positive controlsubstance, has been clinically used as a therapeutic agent for pulmonaryhypertension; and the efficacy thereof has been confirmed in theMCT-induced heart failure model. However, these results are based onrepeated oral administration immediately after the MCT administration.This time, a significant life-prolonging effect could not be observed inthe administration treatment 7 days after the MCT administration. Incontrast, the final survival rate of the group in which compound (A) wasrepeatedly administered at 3 mg/kg twice daily (Group 2) was 50%, whichshowed a significant life-prolonging effect compared with the controlgroup (Group 1). Additionally, the final survival rate of the group inwhich Formulation 21 (ONO-1301Lipo) was intermittently administeredintravenously at 1 mg/kg once weekly (Group 4) was 50%, which alsoshowed a life-prolonging effect comparable with Compound (A) (Group 2).

The group in which Formulation 21 was intermittently administeredintravenously at 1 mg/kg/week (Group 4) showed a life-prolonging effectsimilar to that of the group in which compound (A) was repeatedly orallyadministered at 3 mg/kg twice daily (Group 2), with the total doseamount of Group 4 being 1/42 of that of Group 2; thus, Group 4 wasconfirmed to exhibit a DDS effect as a liposomal formulation.

In contrast, the group in which the ONO-1301 drug substance wasintermittently administered intravenously at 1 mg/kg/week (Group 5)showed no effect (Table 20).

The above results revealed that the repeated oral administration ofcompound (A) (ONO-1301) and the intermittent intravenous administrationof Formulation 21 showed a significant life-prolonging effect bytherapeutic administration to the severe heart failure model after theonset of heart failure (7 days after the MCT administration). Further, asimilar life-prolonging effect was exhibited at a total dose amount of1/42, showing a DDS effect as a stealth liposomal formulation.

2) Analysis of DDS Effect of Formulation 25 (ONO-1301Lipo) on BLMPulmonary Fibrosis Model Mice by Various Administration Methods

1. Method

C57BL/6NCr female mice (7 weeks old) were anesthetized with sodiumpentobarbital, and then intratracheally (intrapulmonary) administeredwith 20 μL of a bleomycin hydrochloride aqueous solution (BLM) twice (40μL in total per animal). In a normal group (Normal), vehicle(physiological saline) was similarly intratracheally administered.

From 6 days to 28 days after the BLM administration, the test substancewas administered to compare the survival rates. As the test substances,compound (A) (ONO-1301) was repeatedly orally administration twicedaily, compound (A) (ONO-1301) was intravenously administered onceweekly, and compound (A) (ONO-1301) was intratracheally administeredonce weekly. Further, Formulation 25 (ONO-1301Lipo) was intravenouslyadministered once weekly, and Formulation 25 (ONO-1301Lipo) wasintratracheally administered once weekly to analyze the DDS effect ofFormulation 25 (ONO-1301Lipo) by comparing the survival rates.

2. Table 21 Shows the Test Group Constitution.

TABLE 21 Number Route of of Group Test group Dose administration animals1 Control 0.5% CMC-Na Oral 10 (vehicle) aqueous solution ×administration twice/day 2 Compound (A) 3 mg/kg × Oral 10 (ONO-1301)twice/day administration 3 Compound (A) 3 mg/kg × Intravenous 10(ONO-1301) once/week administration 4 Formulation 25 1 mg/kg ×Intravenous 10 (ONO- once/week* administration 1301LipoNS) 5 Formulation25 3 mg/kg × Intravenous 10 (ONO- once/week* administration 1301LipoNS)6 Compound (A) 1 mg/kg × Intratracheal 10 (ONO-1301) once/weekadministration 7 Formulation 25 0.3 mg/kg × Intratracheal 10 (ONO-once/week* administration 1301LipoNS) 8 Formulation 25 1 mg/kg ×Intratracheal 10 (ONO- once/week* administration 1301LipoNS) 9 NormalPhysiological Intratracheal 5 saline × once/week administration *Thedose is in terms of compound (A) (ONO-1301).

3. Test Substance Administration

1) Oral Administration: Administration of Vehicle (0.5% CMC-Na AqueousSolution) and Compound (A) (Groups 1 and 2)

From 6 days after the preparation of the lung injury model, the vehicleand compound (A) (3 mg/kg) were repeatedly orally administered twicedaily for 23 days (the administration interval between morning andafternoon was 8 hours or more).

Dose amount: 5 mL/kg×twice/day

Administration method: Gavage oral administration using a disposablepolypropylene syringe and a mouse stomach tube

2) Intravenous Administration: Administration of Compound (A) andFormulation 25 (Groups 3, 4, and 5)

The administration was performed 6 days after the preparation of thelung injury model; after that, tail vein intravenous administration wasperformed once weekly (4 times in total, i.e., 6, 13, 20, and 27 or 28days after the preparation of the model).

Dose amount: 1.5 mL/kg

Administration method: Intravenous administration using a glass syringeand a disposable injection needle 30G

3) Intratracheal Administration: Administration of Compound (A) andFormulation 25 (Groups 6, 7, 8, and 9)

The administration was performed 6 days after the preparation of thelung injury model; after that, intratracheal administration wasperformed once weekly (4 times in total, i.e., 6, 13, 20, and 27 or 28days after the preparation of the model). In normal Group 9 (Normal),physiological saline was administered in a similar manner.

Dose amount: 0.5 mL/kg

Administration method: After anesthesia with pentobarbital (30 to 35mg/kg, i.p.), intratracheal (intrapulmonary) administration wasperformed.

4. Results

1) FIGS. 28 to 30 Show the Results of the Survival Rates.

In the normal group, no deaths were observed. In contrast, the group inwhich the vehicle was orally administered to the lung injury modelshowed a survival rate of 30%. The group in which compound (A) wasorally administered at 3 mg/kg showed a survival rate of 60%, thusachieving a life-prolonging effect compared with the vehicleadministration group. The group in which compound (A) was intravenouslyadministered at 3 mg/kg showed a survival rate of 40%. The groups inwhich Formulation 25 was intravenously administered at 1 mg/kg or 3mg/kg both showed a survival rate of 50%. The group in which compound(A) was intratracheally administered at 1 mg/kg showed a survival rateof 20%. The groups in which Formulation 25 was intratracheallyadministered at 0.3 mg/kg and 1 mg/kg respectively showed survival ratesof 50% and 40%, which are higher than that of the vehicle administrationgroup or the group in which compound (A) was intratracheal administeredat 1 mg/kg.

2) Comparison of Survival Rates

(1) FIG. 29: Comparison with Intermittent Intravenous Administration ofFormulation 25 (ONO-1301Lipo)

In Group 2, the total dose amount of compound (A) (ONO-1301) (3mg/kg×twice×22 days) was 132 mg/kg.

Further, the total dose amount of intravenous administration of compound(A) (ONO-1301) at 3 mg/kg once weekly (Group 3) was 12 mg/kg, and thetotal dose amount of intravenous administration of Formulation 25 at 1mg/kg once weekly (Group 4) was 4 mg/kg. The total dose amount ofintravenous administration of Formulations 25 at 3 mg/kg once weekly(Group 5) was 12 mg/kg.

The survival rate in the vehicle group (Group 1) was 30%. In comparisonwith the group in which compound (A) (ONO-1301) was orally administered(Group 2: 60%), the survival rates of Group 4 (dose amount: 1/33 of thetotal dose amount of Group 2) and Group 5 (dose amount: 1/11 of thetotal dose amount of Group 2) were slightly lower than that of Group 2,and both showed the same value (50%). In contrast, the group in whichthe compound (A) (ONO-1301) drug substance was intravenouslyadministered at 3 mg/kg once weekly (Group 5) showed an even lowersurvival rate (40%) at a dose amount 1/11 that of Group 2.

These results revealed that the intravenous administration ofFormulation 25 once weekly (Groups 4 and 5) to the lung injury modelshowed a prolonging effect on the survival rate, compared withintravenous administration of the compound (A) (ONO-1301) drug substanceonce weekly (Group 3). Further, Groups 4 and 5 showed a slightly lowersurvival rate than that of the group in which the compound (A)(ONO-1301) drug substance was repeatedly orally administered twice daily(Group 2), although the total dose amounts of Groups 4 and 5 were as lowas 1/11 to 1/33 that of Group 2. These results suggested that theintravenous administration of Formulation 25 exhibited a DDS effectspecific to the lung disease site.

(2) FIG. 30: Comparison with Intermittent Intratracheal Administrationof Formulation 25 (ONO-1301Lipo)

In Group 2, the total dose amount of compound (A) (ONO-1301) (3mg/kg×twice×22 days) was 132 mg/kg.

The total dose amount of intratracheal administration of compound (A)(ONO-1301) at 1 mg/kg once weekly (Group 6) was 4 mg/kg, and the totaldose amount of intratracheal administration of Formulation 25 at 0.3mg/kg once weekly (Group 7) was 1.2 mg/kg. The total dose amount ofintratracheal administration of Formulation 25 at 1 mg/kg once weekly(Group 8) was 4 mg/kg.

The survival rate in the vehicle administration group (Group 1) was 30%.In comparison with the group in which compound (A) (ONO-1301) was orallyadministered (Group 2:60%), Group 7 (dose amount: 1/110 of the totaldose amount of Group 2) and Group 8 (dose amount: 1/33 of the total doseof Group 2) showed survival rates of 50% and 40%, respectively.

In contrast, the group in which the compound (A) (ONO-1301) drugsubstance was intravenously administered at 1 mg/kg once weekly (Group6) showed an even lower survival rate (20%) at a dose amount of 1/33that of Group 2.

These results revealed that intratracheal administration of Formulation25 once weekly (Group 7 and Group 8) to the lung injury model showed aprolonging effect on the survival rate, compared with the intratrachealadministration of the compound (A) (ONO-1301) drug substance once weekly(Group 6). Further, Group 7 and Group 8 showed a slightly lower survivalrate than that of the group of repeated oral administration of thecompound (A) (ONO-1301) drug substance twice daily (Group 2), althoughthe total dose amounts of Group 7 and Group 8 were as low as 1/110 to1/33 that of Group 2. These results suggested that the intratrachealadministration of Formulation 25 exhibited a DDS effect specific to thelung disease site. Further, intratracheal administration of Formulation25 at 0.3 mg/kg once weekly showed the same survival rate (50%) as thoseof the intravenous administration of Formulation 25 at 1 mg/kg or 3mg/kg once weekly. This suggested that intratracheal administrationexhibits a DDS effect greater than that of the intravenousadministration.

Based on the above results, the survival rates of the groups in whichFormulation 25 was intermittently administered intravenously once weekly(Groups 4 and 5) and the groups in which Formulation 25 wasintermittently administered intratracheally once weekly (Groups 7 and 8)were compared with that of the vehicle administration group (Group 1),using the bleomycin-induced lung injury model mice from day 6 of thebleomycin administration. As a positive control, a group in whichcompound (A) (ONO-1301) was repeatedly orally administered twice daily(Group 2) was created. Further, a group in which compound (A) (ONO-1301)was intermittently administered intravenously once weekly (Group 3) anda group in which compound (A) (ONO-1301) was intermittently administeredintratracheally (Group 6) were created to compare the DDS effect asONO-1301 liposomal formulations.

In the test, the group in which compound (A) (ONO-1301) was repeatedlyorally administered at 3 mg/kg twice daily from day 6 of the bleomycinadministration showed a prolonged survival rate, suggesting atherapeutic effect.

The intravenous administration of Formulation 25 once weekly (Groups 4and 5) showed a prolonging effect on the survival rate, compared withintravenous administration of the compound (A) (ONO-1301) drug substanceonce weekly (Group 3). Further, Groups 4 and 5 showed a slightly lowersurvival rate than that of the group of repeated oral administration ofthe compound (A) (ONO-1301) drug substance twice daily (Group 2),although the total dose amounts of Groups 4 and 5 were as low as 1/11 to1/33 that of Group 2. These results suggested that intravenousadministration of Formulation 25 showed a DDS effect specific to thelung disease site.

The group in which Formulation 25 was intratracheally administered onceweekly (Groups 7 and 8) showed a prolonging effect on the survival rate,compared with the group in which the compound (A) (ONO-1301) drugsubstance was intratracheally administered once weekly (Group 6).Further, Groups 7 and 8 showed a slightly lower survival rate than thatof the group in which the compound (A) (ONO-1301) drug substance wasrepeatedly orally administered twice daily (Group 2), although the totaldose amount was as low as 1/110 to 1/33 that of Group 2. These resultssuggested that intratracheal administration of Formulation 25 showed aDDS effect specific to the lung disease site. Further, intratrachealadministration of Formulation 25 at 0.3 mg/kg once weekly showed thesame survival rate (50%) as those of intravenous administration ofFormulation 25 at 1 mg/kg or 3 mg/kg once weekly, suggesting that theintratracheal administration achieves a greater DDS effect, i.e., about10 times that of the intravenous administration.

3) Analysis of Effect of Formulation 25 (ONO-1301Lipo) on SpontaneousDilated Cardiomyopathy (J2N-k) Hamsters

(1) Test System

Improvement in the DDS cardiac function in the spontaneous dilatedcardiomyopathy (J2N-k) hamster model by intermittent intravenousadministration of Formulation 25 and compound (A) (ONO-1301) once everytwo weeks was evaluated by comparing the left ventricular ejectionfraction (hereinafter referred to as “EF %”), left ventricularfractional shortening (hereinafter referred to as “% FS”), andhistological evaluation, based on echocardiography, taken as indices. Asa positive control, a group in which compound (A) (ONO-1301) wasrepeatedly orally administered twice daily was created.

As a test substance, Formulation 25 was used by suspending and dilutingwith physiological saline. Compound (A) (ONO-1301) as a control was usedby dissolving in an equivalent amount of aqueous NaOH, and diluting withphysiological saline. For groups of oral administration of compound (A)(ONO-1301), administration was performed as a suspension in a 0.5%CMC-Na aqueous solution.

The spontaneous dilated cardiomyopathy (J2N-k) male hamsters, 20 weeksold at the start of administration, Japan SLC, Inc., were administeredwith the test substances for 8 weeks, and used for comparison.

(2) Table 22 Shows the Group Constitution.

TABLE 22 Number of Group Route of administration animals Group 1;Control Once/2 weeks Intravenous 4 (physiological saline) administrationGroup 2; Formulation 25 *3 Once/2 weeks Intravenous 5 mg/kg(ONO-1301Lipo) administration Group 3; Compound (A) Once/2 weeksIntravenous 4 (ONO-1301): 3 mg/kg administration Group 4; Compound (A)Twice/day Repeated oral 5 (ONO-1301): 3 mg/kg administration *The doseis in terms of compound (A) (ONO-1301).

Liquid dose: 5 mL/kg

(3) Echocardiography

Animals arrived at the age of 18 weeks were subjected to a 2-weekquarantine/acclimation period, and then to the test at the time ofgrouping (test start date) at the age of 20 weeks. Thereafter, testswere conducted 4 and 8 weeks after the test start date, and dissectionwas performed 8 weeks after the test start date.

(4) Dissection and Treatment of Removed Tissue

The final echocardiography was performed 8 weeks after the start ofadministration. Thereafter, dissection was performed.

Dissection was performed as follows. After all of the blood wascollected from the abdominal aorta, and the hamsters were euthanizedunder isoflurane anesthesia, the heart and lungs were removed andweighed. After the weights thereof were measured, the removed parts,including the left and right ventricles, were divided into three parts;i.e., apical, middle, and basal parts, at intervals of about 2 mm alongthe short axis.

For the three divided tissues, the short-axis sections including theright and left ventricles of the middle part were immersed in 4%paraformaldehyde (for general pathological examination), and stored.

(5) Measurement of Left Ventricular Wall Thickness and Area inShort-Axis Sections of Removed Heart

When removed, the heart, including the left and right ventricles, wasdivided into three parts; i.e., apical, middle, and basal parts, atintervals of about 2 mm along the short axis. Among them, one section ofthe middle part was photographed, and the wall thickness at theanterior, lateral, posterior, and septum parts, and the left ventriculararea were measured using image-editing software. For the wall thicknessof the left ventricle, image-editing software was used, the number ofpixels in the scale area of 1 mm² on the photograph was obtained, andthe outer diameter and inner diameter of the entire left ventricle wallwere traced to obtain each number of pixels to thus calculate the leftventricular area by ((the number of pixels of the outer diameter−thenumber of pixels of the inner diameter)/1 mm²). Thereafter, the wallthickness of each portion was measured.

FIG. 31 shows the measurement method. The section was divided into threeparts; i.e., apical, middle, and basal areas, at intervals of about 2mm. Thereafter, using one section on the middle side, the wall thicknessat the anterior, lateral, posterior, and septum parts, and the leftventricular area were measured. For the wall thickness of the leftventricle, image-editing software was used, the number of pixels in thescale area of 1 mm² on the photograph was obtained, and the outerdiameter and inner diameter of the entire left ventricle wall weretraced to obtain each number of pixels to thus calculate the leftventricular wall area by ((the number of pixels of the outerdiameter−the number of pixels of the inner diameter)/1 mm²). Thereafter,the wall thickness of each portion was measured.

(6) Test Results

Table 23 shows the measurement results of EF % values and % FS valuesdetermined by echocardiography.

TABLE 23 EF % % FS Drugs N Pre 4 W 8 W Pre 4 W 8 W Group 1; Control 442.5 ± 3.2 33.6 ± 12.3 31.5 ± 3.5^(a ) 17.8 ± 1.4 13.3 ± 6.1 12.6 ±1.6^(a) Group 4; Compound (A) 5 43.0 ± 7.8 40.4 ± 14.9 40.3 ± 9.2* 18.2± 3.9 17.2 ± 7.2  17.1 ± 4.5* (ONO-1301): 3.0 mg/kg, p.o. Group 3;compound (A) 4 38.8 ± 4.3 35.7 ± 7.4  30.8 ± 2.9^(a ) 16.0 ± 2.1 16.1 ±3.1 12.3 ± 1.3^(a) (ONO-1301): 3.0 mg/kg, intravenous administrationGroup 2; formulation 25: 5 40.5 ± 5.1 44.5 ± 9.9  39.6 ± 4.1* 16.9 ± 2.618.5 ± 4.9   16.5 ± 2.1** 3.0 mg/kg i.v. Data shown: the mean ± standarddeviation *p < 0.05, **p < 0.01 (significantly different from thecontrol value by Student's t-test) ^(a)p < 0.01 (significantly differentfrom the pre-value by Student's t-test)

Echocardiography was performed at the time of grouping, and at week 4and week 8 after the start of administration. The measurement wasperformed 3 times for each individual, and the average value of themeasured data was referred to as the measurement results.

In the control group (Group 1), the EF % values at the time of grouping,and at week 4 and week 8 were 42.5±3.2%, 33.6±12.3%, and 31.5±3.5%,respectively. The EF % value at week 8 showed a significant decrease(p<0.05) compared with the EF % value at the time of grouping. The % FSvalues at the time of grouping and at week 4 and week 8 were17.8±1.4%,13.3±6.1%, and 12.6±1.6%, respectively. Similar to the EF %value, the EF % value at week 8 showed a significant decrease (p<0.01)compared with the % FS value at the time of grouping.

The EF % values of the group of repeat oral administration of compound(A) (ONO-1301) at 3.0 mg/kg×twice/day (Group 4) at the time of groupingand at week 4 and week 8 were 43.0±7.8%, 40.4±14.9%, and 40.3±9.2%,respectively. Compared with the value at the time of grouping, thevalues at week 4 and week 8 showed no significant decrease in cardiacfunction. The EF % value at week 8 was higher than that of the controlgroup, showing a significant difference (p<0.05). The % FS values at thetime of grouping and at week 4 and week 8 were 18.2±3.9%, 17.2±7.2%, and17.1±4.5%, respectively, which were similar to the EF % values.

The EF % values of the group of intravenous administration of compound(A) (ONO-1301) at 3.0 mg/kg (Group 3) at the time of grouping and atweek 4 and week 8 were 38.8±4.3%, 35.7±7.4%, and 30.8±2.9%,respectively. The decrease at week 4 was slower than that of the controlgroup; however, the decrease at week 8 was similar to that of thecontrol group. The EF % value at week 8 showed a significant decrease(p<0.01) from the value at the time of grouping. The % FS values at thetime of grouping and at week 4 and week 8 were 16.0±2.1%, 16.1±3.1%, and12.3±1.3%, respectively. These results were similar to the EF % values,showing no efficacy.

In contrast, the EF % value of the group of intravenous administrationof Formulation 25 (ONO-1301Lipo) at 3.0 mg/kg (Group 2) at the time ofgrouping and at week 4 and week 8 were 40.5±5.1%, 44.5±9.9%, and39.6±4.1%, respectively. Although the difference was not significant, aslight increase was observed at week 4. At week 8, the decrease wassuppressed in a manner similar to that of the group of oraladministration of compound (A) (ONO-1301) at 3.0 mg/kg, and the valuewas significantly higher (p<0.05) compared with that of the controlgroup. The % FS values at the time of grouping and at week 4 and week 8were 16.9±2.6%,18.5±4.9%, and 16.5±2.1%, respectively; thus, the changewas similar to that of the EF % values. The value at week 8 showed asignificant effect (p<0.01) compared with that of the control group(Group 1), and this effect was similar to that of the group of repeatoral administration of compound (A) (ONO-1301) (Group 4).

2. Measurement Results of the Wall Thickness of Removed Heart(Evaluation on the Short-Axis Section of the Middle Part)

Table 24 shows the wall thickness measurement results.

As shown in Table 24, the wall thicknesses of the apical, lateral,posterior, and septum of the middle section in the control group(Group 1) were 0.8±0.1 mm, 1.1±0.2 mm, 1.1±0.2 mm, and 1.0±0.2 mm,respectively.

In the group of oral administration of compound (A) (ONO-1301) at 3.0mg/kg (Group 4), the wall thicknesses of the apical, lateral, posterior,and septum of the middle section were 1.4±0.5 mm, 1.5±0.2 mm, 1.2±0.4mm, and 1.5±0.3 mm, respectively. Thus, the thicknesses of the apical,lateral, and septum were significantly higher (p<0.05) than those of thecontrol group.

In the group of intravenous administration of compound (A) (ONO-1301) at3.0 mg/kg (Group 3), these values were 1.1±0.6 mm, 1.2±0.3 mm, 1.2±0.2mm, and 1.0±0.3 mm, respectively. These measurement results were similarto those of the control group.

In contrast, in the group of intravenous administration of Formulation25 (ONO-1301Lipo) at 3.0 mg/kg (Group 2), these values were 1.7±0.4mm**, 1.5±0.1 mm*, 1.5±0.6 mm^(†), and 1.2±0.3 mm, respectively. Thesevalues were significantly higher and showed a tendency to achieve highervalues in the apical (p<0.01), lateral (p<0.05), and posterior(0.05<p<0.1), compared with those of the control group.

TABLE 24 Body weight (g) Before drug Body weight Left ventricle wallthickness (mm) Drugs N administration at dissection Anterior LateralPosterior Septum Group 1: Control 4 120.2 ± 7.3  129.4 ± 6.7  0.8 ± 0.11.1 ± 0.2  1.1 ± 0.2 1.0 ± 0.2 Group 4: compound (A) 5 118.8 ± 9.6 128.6 ± 3.0   1.4 ± 0.5* 1.5 ± 0.2* 1.2 ± 0.4  1.5 ± 0.3* (ONO-1301):3.0 mg/kg, p.o. Group 3: compound (A) 4 128.7 ± 10.4 144.4 ± 13.3 1.1 ±0.6 1.2 ± 0.3  1.2 ± 0.2 1.0 + 0.3 (ONO-1301): 3.0 mg/kg, intravenousadministration Group 2; Formulation 25: 5 119.6 ± 10.5 138.8 ± 12.4  1.7± 0.4** 1.5 ± 0.1*  1.5 ± 0.6^(†) 1.2 ± 0.3 3.0 mg/kg, intravenousadministration Data shown: the mean ± standard deviation ^(†)0.05 < p <0.1, *p < 0.05, **p < 0.01 (significantly different from the controlvalue by Student's t-test)

3. Measurement Results of the Left Ventricular Wall Area of RemovedHeart (Evaluation on the Short-Axis Section of the Middle Part)

Table 25 shows the left ventricular area measurement results.

As shown in Table 25, the measurement results of the left ventricularwall area of the control group (Group 1), the group of oraladministration of compound (A) (ONO-1301) at 3.0 mg/kg (Group 4), thegroup of intravenous administration of compound (A) (ONO-1301) at 3.0mg/kg (Group 3), and the group of intravenous administration ofFormulation 25 (ONO-1301Lipo) at 3.0 mg/kg (Group 2) were 23.4±5.4 mm²,26.4±4.3 mm², 23.9±4.5 mm², and 29.5±6.4 mm², respectively. Thesemeasurement results showed no significant difference; however,reflecting the wall thickness measurement results, there was a tendencythat the control group (Group 1) and the group of intravenousadministration of compound (A) (ONO-1301) at 3.0 mg/kg (Group 3) showedlower values, while the group of oral administration of compound (A)(ONO-1301) at 3.0 mg/kg (Group 4) and the group of intravenousadministration of Formulation 25 (ONO-1301Lipo) at 3.0 mg/kg (Group 4)showed higher values.

TABLE 25 Left ventricular area (Middle part) Drugs N (mm²) Group 1:Control 4 23.4 ± 5.4 Group 4: Compound (A) (ONO- 5 26.4 ± 4.3 1301): 3.0mg/kg, p.o. Group 3: Compound (A) (ONO- 4 23.9 ± 4.5 1301): 3.0 mg/kg,i.v. Group 2; Formulation 25; 5  29.5 ± 6.4^(†) 3.0 mg/kg, i.v. Datashown: the mean ± standard deviation 0.05 < p < 0.1 (significantlydifferent from the control value by Student's t-test)

As described above, the cardiac function improvement effect ofFormulation 25 (ONO-1301Lipo) was analyzed using spontaneous dilatedcardiomyopathy (J2N-k) hamsters. The test was initiated after the onsetof the pathological condition, and at the age of 20 weeks when thecardiac function considerably decreased. The test substance wasadministered until week 28, and the change in the cardiac function wasanalyzed.

In the cardiac function evaluation using EF % and % FS values determinedby echocardiography as indices, the group in which compound (A)(ONO-1301) was repeatedly orally administered at 3.0 mg/kg×twice/day(Group 4) and the group in which Formulation 25 (ONO-1301Lipo) wasintermittently administered intravenously at 3.0 mg/kg 4 times in total(at the time of grouping; and at week 2, week 4, and week 6) (Group 2)showed an effect in terms of suppressing a decrease in the cardiacfunction, compared with the control group (Group 1) and the group ofintravenous administration of compound (A) (ONO-1301) at 3.0 mg/kg(Group 3). In the measurement results of the left ventricle wallthickness, the control group (Group 1) and the group of intravenousadministration of compound (A) (ONO-1301) at 3.0 mg/kg (Group 3) showedlow values, which suggested the progress of thinning. In contrast, thegroup of repeat oral administration of compound (A) (ONO-1301) at 3.0mg/kg (Group 4) and the group of Formulation 25 (ONO-1301Lipo) at 3.0mg/kg (Group 2) showed higher values, suggesting the suppression of theprogress of thinning, compared with the control group (Group 1) and thegroup of intravenous administration of compound (A) (ONO-1301) at 3.0mg/kg (Group 3). This is assumed to reflect the results of EF % and % FSvalues determined by echocardiography.

The above results suggested that the group of repeat oral administrationof compound (A) (ONO-1301) at 3.0 mg/kg×twice/day (Group 4) and thegroup of intermittent intravenous administration of Formulation 25(ONO-1301LipoNS) at 3.0 mg/kg once every 2 weeks (Group 2) suppressed adecrease in the cardiac function in the spontaneous dilatedcardiomyopathy (J2N-k) hamsters.

The total dose amount of the repeat oral administration of compound (A)(ONO-1301) at 3 mg/kg×twice×56 days was 336 mg/kg, while the total doseamount of the intravenous administration of Formulations 25(ONO-1301Lipo) at 3 mg/kg×4 times was 12 mg/kg; both of these showed asimilar efficacy. The intermittent intravenous administration ofcompound (A) (ONO-1301) at 3 mg/kg once every 2 weeks showed no effect.Accordingly, the intermittent intravenous administration of Formulation25 (ONO-1301Lipo) once every 2 weeks showed the effect at a dose of 1/28of that of the repeat oral administration of compound (A) (ONO-1301).This confirmed the DDS effect of intravenous administration ofFormulation 25 (ONO-1301Lipo).

Tables 26 and 27 show the body weight change at the time of grouping andat the time of dissection, and the weights of heart and lung at the timeof dissection. No change was observed in any of these.

TABLE 26 Body weight (g) Before drug Body weight at Drugs Nadministration dissection Control 4 120.2 ± 7.3  129.4 ± 6.7  compound(A) (ONO-1301): 5 118.8 ± 9.6  128.6 ± 3.0  3.0 mg/kg, p.o. Compound (A)(ONO-1301): 4 128.7 ± 10.4 144.4 ± 13.3 3.0 mg/kg, i.v. Formulation 25:5 119.6 ± 10.5 138.8 ± 12.4 3.0 mg/kg, i.v. Data shown: the mean ±standard deviation

TABLE 27 Body weight (g) Body Tissue weight per 100 g weight at Realtissue weight (g) of the body weight (g) Drugs N dissection Heart LungHeart Lung Control 4 129.4 ± 6.7  0.4674 ± 0.0666 0.5640 ± 0.0949 0.3602± 0.0340 0.4346 ± 0.0548 Compound (A) 5 128.6 ± 3.0  0.4379 ± 0.01360.5202 ± 0.0336 0.3404 ± 0.0032 0.4043 ± 0.0203 (ONO-1301): 3.0 mg/kg,p.o. Compound (A) 4 144.4 ± 13.3 0.5028 ± 0.0631 0.5740 ± 0.0527 0.3475± 0.0165 0.3998 ± 0.0470 (ONO-1301): 3.0 mg/kg, i.v. Formulation 25: 5138.8 ± 12.4 0.4797 ± 0.0673 0.5645 ± 0.0625 0.3446 ± 0.0222 0.4068 ±0.0276 3.0 mg/kg, i.v. Data shown: the mean ± standard deviation

4) Analysis of Effect of Single Intravenous Administration ofFormulation 25 (ONO-1301LipoNS) on Rat Model of Ischemia by CompleteCoronary Artery Ligation

An improvement in the cardiac function against ischemic heart diseaseand an effect of preventing death from heart failure by intermittentintravenous administration of Formulation 25 (ONO-1301Lipo) or compound(A) (ONO-1301) were analyzed by using the left ventricular ejectionfraction (hereinafter referred to as “EF %”), left ventricularfractional shortening (hereinafter referred to as “% FS”), andhistological evaluation, based on echocardiography, as indices.

(1) Preparation of Myocardial Ischemia Model

Male Sprague-Dawley rats (CLEA Japan, Inc., 6 weeks old when arrived)were anesthetized with a liquid mixture of 0.5 mg/kg of midazolam(Dormicum Injection 10 mg, Astellas Pharma Inc.) and 2 mg/kg of xylazine(Celactal 2% injection, Bayer Japan Ltd.). Thereafter, the animal tailveins were secured, and Propofol (1% Diprivan Injection; AstraZeneca KK)was continuously infused at 6 to 10 mg/kg/hr with a syringe pump(Terufusion TE-3310N; Terumo Corporation) to maintain deep anesthesia.Thereafter, a tracheal cannula (a cut-down catheter with an outsidediameter of 2.0 mm, produced by JMS Co., Ltd.) was inserted andindwelled, and a respirator (for small animals; Shinano Seisakusho) wasconnected to maintain breathing at a stroke volume of 1 mL/100 g/stroke(±1 mL) and a stroke rate of 70 times/min (±10 times). Thereafter, theanimals were fixed in a recumbent position from a dorsal fixed positionso that the left chest faced upward, and the epidermis and muscle layerbetween the third to fourth or fourth to fifth ribs were incised with asurgical knife. After confirming that the knife reached the thoraciccavity, the intercostal space was widened with a rib spreader, and astate in which the anterior descending branch (hereinafter referred toas “LAD”) from the left atrial appendage was directly visible at thefront was secured.

Thereafter, the translucent thin pericardium was removed with tweezersto expose the myocardium. Thereafter, the LAD located at the margin ofthe left atrial appendage was pierced and secured with a nylon threadwith a 6-0 or 7-0 needle at a depth of 2 to 3 mm using a microneedleholder, and the LAD was completely ligated. Thereafter, the muscle layerand epidermis were sutured and closed with a 4-0 or 5-0 nylon thread.After the chest was closed, 50 mg of cefamedin was subcutaneouslyadministered to the treatment site, and the treatment was terminated.

(2) Table 28 Shows the Group Constitution.

TABLE 28 Route of Number of Group administration animals Group 1: Normal— 3 Group 2: Control Intravenous 5 (Physiological saline) administrationGroup 3: Formulation 25 Intravenous 5 (ONO-1301Lipo): 0.3 mg/kgadministration Group 4: Formulation 25 Intravenous 5 (ONO-1301Lipo): 1.0mg/kg administration Group 5: Formulation 25 Intravenous 5(ONO-1301Lipo): 3.0 mg/kg administration Group 6: Compound (A)Intravenous 5 (ONO-1301): 3.0 mg/kg administration 1) Liquid dose: 5mL/kg 2) Single intravenous administration to the tail 24 hours afterinfarction

(3) Echocardiography

Echocardiography was performed to confirm the cardiac function improvingeffect of the test substances using the left ventricular ejectionfraction (hereinafter referred to as “EF %”) as an index.

The test was performed 23 hours after the preparation of the model (testfor grouping), the animals whose EF % value decreased by 25% or more ofthat of normal animals were selected, grouping was performed, and asolution for administration of the test substances was administered bytail vein intravenous administration 24 hours after the preparation ofthe model. Thereafter, echocardiography was performed 7 and 14 daysafter the test substance administration.

(4) Dissection and Treatment of Removed Tissue

Dissection was performed after the completion of the echocardiography atday 14 after the administration of the test substance. For dissection,all of the blood was collected from the abdominal aorta, and the ratswere euthanized under isoflurane anesthesia; thereafter, the heart wasremoved and weighed. After measuring the heart weight, the infarct area,including the left and right ventricles, was divided into three partsalong the short axis.

(5) Test Results

(1) Measurement Results of EF % Value Determined by Echocardiography

Tables 29 and 30 show the echocardiography results.

As shown in Table 29, the EF % value in the normal animals was 85.0±1.9%(n=4). The EF % value of each group 23 hours after the complete LADligation and before the test substance administration was such that thecontrol group was 52.8±6.8% (n=5), and the groups of administration ofFormulation 25 (ONO-1301Lipo) at 0.3 mg/kg, 1.0 mg/kg, and 3.0 mg/kgwere 55.1±2.4% (n=5), 55.0±4.1% (n=5), and 54.7±5.2% (n=5),respectively. The group of administration of compound (A) (ONO-1301) at3 mg/kg was 56.7±5.5% (n=5).

The EF % values of all of the groups before the test substanceadministration showed a significant decrease (p<0.01) compared with theEF % value of the normal animals; no significant difference was observedamong the groups.

As shown in Table 29, the change in the EF % value of each group at day7 and day 14 after the administration were as follows. In the controlgroup, the EF % value was 52.8±6.8% (n=5) before the administration; anddecreased in a time-dependent manner to 48.9±4.0% and 39.0±5.2%,respectively. The EF % value 2 weeks later showed a significant decrease(p<0.05) compared with that before the administration.

In the 0.3 mg/kg administration group of Formulation 25(ONO-1301LipoNS), the EF % value was 55.1±2.4% (n=5) before theadministration; and the values at day 7 and day 14 were 50.7±6.2% and42.0±6.2%, respectively, showing similar changes to those of the controlgroup. Similar to the control group, the EF % value of this group at day14 also showed a significant decrease (p<0.05) compared with that beforethe administration.

In the 1.0 mg/kg administration group, the EF % value was 55.0±4.1%(n=5) before the administration; and the values at day 7 and day 14 were53.6±7.5% and 54.7±8.2%, respectively, which were similar to the EF %value before the administration. The EF % value at day 14 showed asignificant increase (p<0.05) compared with that of the control group.

In the 3.0 mg/kg administration group, the EF % value was 54.7±5.2%(n=5) before the administration, and increased to 62.4±8.7% and58.2±13.2%, respectively. The increase in the EF % value showed a peakon day 7 after the administration compared with the EF % value beforethe administration, indicating an improvement in the cardiac function.The EF % values at day 7 and day 14 after the test substanceadministration showed a significant suppression of a decrease (p<0.05and p<0.01, respectively) compared with those of the control group.

In the group of administration of compound (A) (ONO-1301) at 3.0 mg/kg,the EF % value of was 56.7±5.5% (n=5) before the administration, andchanged to 54.6±8.8% and 53.2±7.1%, respectively, showing a tendency ofa decrease from the EF % value before the administration, although thedifference was not significant. However, a comparison with those of thecontrol group at day 7 and day 14 revealed no significant difference inboth day 7 and day 14.

4. Measurement Results of % FS Value Determined by Echocardiography

As shown in Table 29, the % FS value in the normal animals was 49.4±2.4%(n=4). The % FS value of each group 23 hours after the complete LADligation and before the test substance administration was such that thecontrol group was 23.9±3.7% (n=5); and the groups of administration ofFormulation 25 (ONO-1301Lipo) at 0.3 mg/kg, 1.0 mg/kg, and 3.0 mg/kgwere 25.0±1.7% (n=5), 25.8±2.4% (n=5), and 25.0±3.0% (n=5),respectively. The % FS value of the group of administration of compound(A) (ONO-1301) at 3 mg/kg was 26.5±3.7% (n=5). Usually, the normal % FSvalue is clinically said to be 28% or more. Although the difference wasslight, the % FS values of all of the groups were lower than this value.

The % FS values of all of the groups before the test substanceadministration showed a significant decrease (p<0.01) compared with theEF % value of the normal animals; no significant difference was observedamong the groups.

As shown in Table 30, the change in the % FS value of each group at day7 and day 14 after the administration were as follows. In the controlgroup, the % FS value was 23.9±3.7% before the administration (n=5); anddecreased in a time-dependent manner to 21.8±2.2% and 16.7±2.7%,respectively. Similar to the EF % value, the % FS value 2 weeks latershowed a significant decrease (p<0.05) compared with that before theadministration.

In the 0.3 mg/kg administration group of Formulation 25 (ONO-1301Lipo),the % FS value was 25.0±1.7% (n=5) before the administration; and thevalues at day 7 and day 14 were 22.9±3.4% and 18.4±3.2%, respectively,showing similar changes to those of the control group. Similar to thecontrol group, the % FS value at day 14 also showed a significantdecrease (p<0.05) compared with that before the administration.

In the 1.0 mg/kg administration group, the % FS value was 25.8±2.4%(n=5) before the administration; and the values at day 7 and day 14 were24.8±4.2% and 25.4±5.0%, respectively, which were similar to the FS %value before the administration.

In the 3.0 mg/kg administration group, the % FS value was 25.0±3.0%(n=5) before the administration, and increased to 30.1±5.4% and27.9±8.5%, respectively. The increase in the % FS value showed a peak onday 7 after the administration compared with the % FS value before theadministration. The % FS values at day 7 and day 14 after the testsubstance administration both showed a significant increase (p<0.05)compared with those of the control group.

In the group of administration of compound (A) (ONO-1301) at 3.0 mg/kg,the % FS value was 26.5±3.7% (n=5) before the administration, andchanged to 25.4±5.6% and 24.5±4.4%, respectively, showing a tendency ofa decrease from the % FS value before the administration, although thedifference was not significant. However, a comparison with those of thecontrol group at day 7 and day 14 revealed no significant difference inboth day 7 and day 14.

As described above, the measurement results showed correlation betweenthe EF % and % FS values, which are used as indices of the evaluation ofthe cardiac function, in each group; and the single intravenousadministration of Formulation 25 (ONO-1301LipoNS) at 1.0 mg/kg or 3.0mg/kg showed a cardiac function improving effect.

TABLE 29 Drugs N EF % % FS Group 1: Normal 4 85.0 ± 1.9  49.4 ± 2.4 Group 2: Control 5 52.8 ± 6.8** 23.9 ± 3.7** Group 3: Formulation 25: 555.1 ± 2.4** 25.0 ± 1.7** 0.3 mg/kg Group 4: Formulation 25: 5 55.0 ±4.1** 25.8 ± 2.4** 1.0 mg/kg Group 5: Formulation 25: 5 54.7 ± 5.2**25.0 ± 3.0** 3.0 mg/kg Group 6: Compound (A) 5 56.7 ± 5.5** 26.5 ± 3.7**(ONO-1301): 3.0 mg/kg Data shown: the mean ± standard deviation **p <0.01 (significantly different from the normal value by Dunnett's test)

TABLE 30 EF % % FS Drugs N Pre 1 W 2 W Pre 1 W 2 W Group 2: Control 552.8 ± 6.8 48.9 ± 4.0 39.0 ± 5.2^(a) 23.9 ± 3.7 21.8 ± 2.2 16.7 ±2.7^(a) Group 3: Formulation 25: 5 55.1 ± 2.4 50.7 ± 6.2 42.0 ± 6.2^(a)25.0 ± 1.7 22.9 ± 3.4 18.4 ± 3.2^(a) 0.3 mg/kg Group 4: Formulation 25:5 55.0 ± 4.1 53.6 ± 7.5  54.7 ± 8.2* 25.8 ± 2.4 24.8 ± 4.2 25.4 ± 5.0 1.0 mg/kg Group 5: Formulation 25: 5 54.7 ± 5.2  62.4 ± 8.7*   58.2 ±13.2** 25.0 ± 3.0  30.1 ± 5.4*  27.9 ± 8.5* 3.0 mg/kg Group 6: Compound(A) 5 56.7 ± 5.5 54.6 ± 8.8 53.2 ± 7.1  26.5 ± 3.7 25.4 ± 5.6 24.5 ±4.4  (ONO-1301): 3.0 mg/kg Data shown: the mean ± standard deviation *p< 0.05, **p < 0.01 (significantly different from the control value byDunnett's test) ^(a)p < 0.05 (significantly different from the pre-valueby t-test)

TABLE 31 (g) Body weight Heart weight Before Total weight per 100 g ofpreparation Before drug At of isolated body weight Drugs N of AMIadministration dissection heart (g) (g) Group 2: Control 5 334.3 ± 19.3315.2 ± 15.2 407.6 ± 14.8 1.3997 ± 0.0876 0.3431 ± 0.0219 Group 3:Formulation 25: 5 331.2 ± 19.7 317.7 ± 16.2 408.2 ± 23.2 1.3191 ± 0.14930.3236 ± 0.0362 0.3 mg/kg Group 4: Formulation 25: 5 340.2 ± 18.7 320.8± 21.5 412.4 ± 13.4 1.3179 ± 0.0655 0.3199 ± 0.0206 1.0 mg/kg Group 5:Formulation 25: 5 329.2 ± 16.1 315.3 ± 14.2 409.8 ± 9.1  1.3426 ± 0.10250.3277 ± 0.0255 3.0 mg/kg Group 6: compound (A) 5 339.7 ± 13.6 325.1 ±22.3 406.7 ± 37.4 1.3312 ± 0.1827 0.3267 ± 0.0235 (ONO-1301): 3.0 mg/kg

Infarct Area Evaluation Method

For the sections, the apical portion was removed, and the middle areawas sectioned into two parts at an interval of about 2 mm. Thereafter,the infarct area of the two sections on the apical and basal sides wasmeasured. The infarct area was evaluated based on the ratio of theinfarct area to the entire left ventricular area, and the ratio of thelength of the normal area or the infarct area to the left ventricularouter diameter.

FIG. 32 shows an infarct area evaluation method. For the sections, theapical portion was removed, and the middle area was sectioned into twoparts at an interval of about 2 mm. Thereafter, the infarct area of thetwo sections on the apical and basal sides was measured. The infarctarea was evaluated based on the ratio of the infarct area to the entireleft ventricular area, and the ratios of the length of the normal areaor the infarct area to the left ventricular outer diameter.

The results revealed that the group of administration of Formulation 25at 3 mg/kg showed a significant decrease in the ratio of the infarctarea relative to the entire left ventricle area, and the ratio of theinfarct area in the left ventricular outer diameter area (Table 32 andTable 33).

The results confirmed that Formulation 25 reduced the infarct area,indicating that an effect was exhibited. In contrast, the administrationof compound (A) (ONO-1301) at 3 mg/kg showed no effect. These resultssuggested that Formulation 25 exhibited a DDS effect.

TABLE 32 Left ventricular area (mm²) Left ventricular area (mm²) NormalInfarct Infarct Normal Infarct Infarct Drugs N region region rate (%)region region rate (%) Group 2: Control 5 35.7 ± 6.13 17.8 ± 3.22 34 ±7.3 41.1 ± 6.80 13.3 ± 2.45 25 ± 5.2 Group 3: Formulation 25: 5 36.7 ±1.43 23.4 ± 8.54 38 ± 8.7  41.8 ± 10.70 18.9 ± 4.56 31 ± 5.7 0.3 mg/kgGroup 4: Formulation 25: 5 34.6 ± 7.73 15.9 ± 2.12 32 ± 6.4 42.0 ± 9.0417.6 ± 2.65 30 ± 6.4 1.0 mg/kg Group 5: Formulation 25: 5 41.0 ± 4.88 6.5 ± 4.83  13 ± 9.8** 49.7 ± 7.72  8.1 ± 3.72  14 ± 6.4* 3.0 mg/kgGroup 6: Compound A: 5 31.1 ± 2.83 18.1 ± 5.18 36 ± 8.0 41.0 ± 8.03 14.6± 3.23 26 ± 2.2 3.0 mg/kg Data shown in the table: the mean ± standarddeviation *p < 0.05; **p < 0.01 (Dunnett's test)

TABLE 33 Left ventricular Left ventricular outer diameter (cm) outerdiameter (cm) Normal Infarct Infarct Normal Infarct Infarct Drugs Nregion region rate (%) region region rate (%) Group 2: Control 5 1.9 ±0.36 1.4 ± 0.18 42 ± 4.9  2.5 ± 0.23 1.2 ± 0.20 32 ± 5.2 Group 3:Formulation 25: 5 1.6 ± 0.34 1.5 ± 0.53 47 ± 14.9 2.3 ± 0.06 1.3 ± 0.1936 ± 3.3 0.3 mg/kg Group 4: Formulation 25: 5 1.9 ± 0.53 1.1 ± 0.24 37 ±12.2 2.3 ± 0.39 1.2 ± 0.31 34 ± 9.5 1.0 mg/kg Group 5: Formulation 25: 52.4 ± 0.16 0.6 ± 0.28  20 ± 7.7** 2.7 ± 0.16 0.7 ± 0.21  19 ± 5.3* 3.0mg/kg Group 6: Compound A: 5 1.7 ± 0.41 1.5 ± 0.15 46 ± 7.5  2.3 ± 0.411.2 ± 0.37  34 ± 10.2 3.0 mg/kg Data shown in the table: the mean ±standard deviation *p < 0.05; **p < 0.01 (Dunnett's test)

The single intravenous administration of Formulation 25 (ONO-1301Lipo)suppressed the onset of the pathological condition in the rat heartischemia model in a dose-correlated manner. Further, the administrationof Formulation 25 at 3 mg/kg showed an improving effect over the Pre.Although the single intravenous administration of the compound (A)(ONO-1301) drug substance at 3 mg/kg showed a suppression effect similarto that of Formulation 25 at 1 mg/kg, no effect was observed in terms ofreducing the infarct area.

These results confirmed that Formulation 25 exhibited a DDS effect.

1. A pharmaceutical composition for disease site-specific treatment,comprising a stealth liposome having a prostaglandin I2 receptor agonistencapsulated therein.
 2. The pharmaceutical composition according toclaim 1, wherein the prostaglandin I2 receptor agonist includes at leasta compound represented by formula (I):

wherein

wherein e represents an integer of 3 to 5, f represents an integer of 1to 3, p represents an integer of 1 to 4, q represents 1 or 2, and rrepresents an integer of 1 to 3; R¹ represents a hydrogen atom or a C₁₋₄alkyl group; R² represents (i) a hydrogen atom, (ii) a C₁₋₈ alkyl group,(iii) a phenyl group or a C₄₋₇ cycloalkyl group, (iv) a 4- to 7-memberedmonocyclic ring containing one nitrogen atom, (v) a C₁₋₄ alkyl groupsubstituted with a benzene ring or a C₄₋₇ cycloalkyl group, or (vi) aC₁₋₄ alkyl group substituted with a 4- to 7-membered monocyclic ringcontaining one nitrogen atom; and R³ represents (i) a C₁₋₈ alkyl group,(ii) a phenyl group or a C₄₋₇ cycloalkyl group, (iii) a 4- to 7-memberedmonocyclic ring containing one nitrogen atom, (iv) a C₁₋₄ alkyl groupsubstituted with a benzene ring or a C₄₋₇ cycloalkyl group, or (v) aC₁₋₄ alkyl group substituted with a 4- to 7-membered monocyclic ringcontaining one nitrogen atom; provided that when

is a group represented by (iii) or (iv), —(C—(CH₂)_(p)— and═CH—(CH₂)_(s)— are bound to position a or b on the ring, and cyclicstructures in R² and R³ are optionally substituted with one to threeC₁₋₄ alkyl groups, C₁₋₄ alkoxy groups, halogen atoms, nitro groups, ortrihalomethyl groups; or a salt thereof.
 3. The pharmaceuticalcomposition according to claim 1, wherein the prostaglandin I2 receptoragonist includes at least the following compound (A): (A)({5-[2-({[(1E)-phenyl(pyridin-3-yl)methylene]amino}oxy)ethyl]-7,8-dihydronaphthalen-1-yl}oxy)aceticacid (ONO-1301) represented by formula (II):

or a salt of compound (A).
 4. The pharmaceutical composition accordingto claim 1, wherein the prostaglandin I2 receptor agonist includes atleast one of the following compounds (B) to (E): (B) sodium(±)-(1R,2R,3aS,8bS)-2,3,3a,8b-tetrahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-hydroxy-4-methyl-1-octen-6-ynyl]-1H-cyclopenta[b]benzofuran-5-butanoate(beraprost), or a derivative thereof that is a carbacyclic PGI2derivative, (C)[4-[(5,6-diphenylpyrazinyl)(1-methylethyl)amino]butoxy]-acetic acid(MRE-269), (D)(2E)-7-{(1R,2R,3R)-3-hydroxy-2-[(1E,3S,5S)-3-hydroxy-5-methylnon-1-en-1-yl]-5-oxycyclopentyl}hept-2-enoicacid (limaprost), ornoprostil, enprostil, or misoprostol; or aderivative of any of these compounds that is a PEF derivative, and (E)2-{4-[(5,6-diphenylpyrazin-2-yl)(propan-2-yl)amino]butoxy}-N-(methanesulfonyl)acetamide(NS-304; selexipag); or a salt of any of compounds (B) to (E).
 5. Thepharmaceutical composition according to claim 1, wherein theprostaglandin I2 receptor agonist includes at least one member selectedfrom the group consisting of ONO-1301, beraprost, limaprost, and NS-304.6. The pharmaceutical composition according to claim 1, wherein thestealth liposome is obtainable by using at least a prostaglandin I2receptor agonist and a phospholipid by the Bangham method, hydrationdispersion method, reverse phase evaporation method, ethanol injectionmethod, ethanol dilution method, homogenization method, mechanochemicalmethod, direct dispersion method, extruder method, French press method,remote loading method, dehydration-rehydration method, freeze-thawmethod, ultrasonic method, or lipid-compound film method; or an improvedmethod of any of these methods.
 7. The pharmaceutical compositionaccording to claim 1, wherein the stealth liposome has an averageparticle size of 50 to 200 nm, and comprises 5 to 50 parts by weight ofthe phospholipid and 0.05 to 5 parts by weight of PEG-modifiedphosphoethanolamine, per part by weight of the prostaglandin I2 receptoragonist.
 8. The pharmaceutical composition according to claim 1, whereinthe stealth liposome comprises 0.05 to 5 parts by weight ofMPEG2000-DSPE per part by weight of the prostaglandin I2 receptoragonist; the prostaglandin I2 receptor agonist includes at least onemember selected from the group consisting of ONO-1301, beraprost,limaprost, and NS-304; and the stealth liposome releases theprostaglandin I2 receptor agonist over a period of 3 hours to 4 weeks.9. The pharmaceutical composition according to claim 1, wherein thestealth liposome comprises a prostaglandin I2 receptor agonist, aphospholipid, a PEG-modified phosphoethanolamine, and a water-miscibleorganic solvent, and does not comprise a sterol; the liposome isobtainable by a production method comprising the following steps (1) to(8): (1) mixing the prostaglandin I2 receptor agonist, the phospholipid,and the PEG-modified phosphoethanolamine in the solvent in amounts suchthat at least 5 mg of the phospholipid and at least 0.05 mg of thePEG-modified phosphoethanolamine are present per mg of the prostaglandinI2 receptor agonist, (2) heating the mixture obtained in step (1) toprepare a melt, (3) instantly freezing the melt obtained in step (2),(4) freeze-drying the frozen product obtained in step (3) to remove thesolvent, (5) heating the freeze-dried product obtained in step (4) todisperse the heated product in an aqueous phosphate buffer solution, (6)sizing the dispersion obtained in step (5) with an extruder, (7)ultrafiltrating the dispersion obtained in step (6) to removeunencapsulated material, and (8) adding a sugar to the dispersionobtained in step (7) and freeze-drying the dispersion; and the liposomecontains at least 0.001 mg of the prostaglandin I2 receptor agonist per1.0 mg of the phospholipid, and has an average particle size of 50 to200 nm.
 10. The pharmaceutical composition according to claim 1, whereinthe composition is for intravenous administration, intracoronaryadministration, inhalation, intramuscular administration, subcutaneousadministration, oral administration, transmucosal administration,transdermal administration, or an internal organ, and is in the form ofan injectable formulation, an oral preparation, an inhalant, anebulizer, an ointment, a patch, or a spray.
 11. The pharmaceuticalcomposition according to claim 1, wherein a single intravenous dose ofthe composition is 0.001 to 100 mg in terms of the prostaglandin I2receptor agonist.
 12. The pharmaceutical composition according to claim11, wherein a disease to be treated with the composition iscardiovascular disease, respiratory disease, urinary disease,gastrointestinal disease, bone disease, neurodegenerative disease,vascular disease, dental disease, eye disease, skin disease, otherinflammatory disease, ischemic organ disorder, diabetic complication,tissue fibrotic disease, tissue degenerative disease, or hair loss; andthe composition comprises a liposome.
 13. The pharmaceutical compositionaccording to claim 11, wherein a disease to be treated with thecomposition is a cardiovascular disease such as ischemic and dilatedcardiomyopathy, atherosclerosis obliterans, vasculitis syndrome,valvular disease, aortic stenosis, chronic heart failure, or diastolicfailure; a respiratory lung disease such as pulmonary hypertension,pulmonary fibrosis, asthma, or chronic obstructive pulmonary disease; agastrointestinal or urinary disease such as chronic kidney disease,chronic hepatitis, or chronic pancreatitis; or a neurodegenerativedisease such as chronic phase of cerebral infarction, Alzheimer'sdisease, diabetic neuropathy, Parkinson's disease, or amyotrophiclateral sclerosis; and the composition comprises a liposome.
 14. Thepharmaceutical composition according to claim 2, wherein the stealthliposome is obtainable by using at least a prostaglandin I2 receptoragonist and a phospholipid by the Bangham method, hydration dispersionmethod, reverse phase evaporation method, ethanol injection method,ethanol dilution method, homogenization method, mechanochemical method,direct dispersion method, extruder method, French press method, remoteloading method, dehydration-rehydration method, freeze-thaw method,ultrasonic method, or lipid-compound film method; or an improved methodof any of these methods.
 15. The pharmaceutical composition according toclaim 3, wherein the stealth liposome is obtainable by using at least aprostaglandin I2 receptor agonist and a phospholipid by the Banghammethod, hydration dispersion method, reverse phase evaporation method,ethanol injection method, ethanol dilution method, homogenizationmethod, mechanochemical method, direct dispersion method, extrudermethod, French press method, remote loading method,dehydration-rehydration method, freeze-thaw method, ultrasonic method,or lipid-compound film method; or an improved method of any of thesemethods.
 16. The pharmaceutical composition according to claim 4,wherein the stealth liposome is obtainable by using at least aprostaglandin I2 receptor agonist and a phospholipid by the Banghammethod, hydration dispersion method, reverse phase evaporation method,ethanol injection method, ethanol dilution method, homogenizationmethod, mechanochemical method, direct dispersion method, extrudermethod, French press method, remote loading method,dehydration-rehydration method, freeze-thaw method, ultrasonic method,or lipid-compound film method; or an improved method of any of thesemethods.
 17. The pharmaceutical composition according to claim 5,wherein the stealth liposome is obtainable by using at least aprostaglandin I2 receptor agonist and a phospholipid by the Banghammethod, hydration dispersion method, reverse phase evaporation method,ethanol injection method, ethanol dilution method, homogenizationmethod, mechanochemical method, direct dispersion method, extrudermethod, French press method, remote loading method,dehydration-rehydration method, freeze-thaw method, ultrasonic method,or lipid-compound film method; or an improved method of any of thesemethods.
 18. The pharmaceutical composition according to claim 2,wherein the stealth liposome has an average particle size of 50 to 200nm, and comprises 5 to 50 parts by weight of the phospholipid and 0.05to 5 parts by weight of PEG-modified phosphoethanolamine, per part byweight of the prostaglandin I2 receptor agonist.
 19. The pharmaceuticalcomposition according to claim 3, wherein the stealth liposome has anaverage particle size of 50 to 200 nm, and comprises 5 to 50 parts byweight of the phospholipid and 0.05 to 5 parts by weight of PEG-modifiedphosphoethanolamine, per part by weight of the prostaglandin I2 receptoragonist.
 20. The pharmaceutical composition according to claim 4,wherein the stealth liposome has an average particle size of 50 to 200nm, and comprises 5 to 50 parts by weight of the phospholipid and 0.05to 5 parts by weight of PEG-modified phosphoethanolamine, per part byweight of the prostaglandin I2 receptor agonist.